University of Southern California Dissertations and Theses

Structure and function of archaeal McM helicase

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Structure and function of archaeal McM helicase

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Content STRUCTURE AND FUNCTION OF ARCHAEAL MCM HELICASE

by

Aaron Samuel Brewster

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)

May 2010

Copyright 2010 Aaron Samuel Brewster
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Dedication

For April
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Acknowledgements
I thank Dr. Xiaojiang Chen for being my advisor, Dr. U. Sen for help with
crystallization and data collection, Dr. R. Zhang at 19-ID beamline in Argonne National
laboratory, and staff at the 8.2.1 beamline at Berkeley’s ALS for assistance with data
collection. I thank Lauren Holden for her assistance in editing Chapter 4, and Drs. D.
Gai, B. Greenleaf, and M. Klein for insightful discussions throughout. This work was
supported in part by NIH grant R01GM080338 to Xiaojiang Chen.

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Table of Contents

Dedication
Acknowledgements
List of Tables
List of Figures
Abstract
Chapter 1: Introduction
Chapter 2: Insights into Helicase Function from the Structure of a Near-Full
Length Archaeal MCM Helicase
Chapter 3: Mutational Analysis of an Archaeal MCM Exterior Hairpin Reveals
Critical Residues for Helicase Activity
Chapter 4: Modeling the DNA unwinding activity of MCM helicase
Bibliography
Appendices:
Appendix A: Cloning and purification of additional MCMs
Appendix B: 14 Additional MCM Mutants
Appendix C: Homology modeling of eukaryotic MCMs
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List of Tables

Table 1: Summary of ssoMCM mutational studies
Table 2: Kinetic parameters of ssoMCM mutants
Table 3: Crystallography Statistics
Table 4: Kinetic Parameters of EXT-hp mutants
Table 5: Summary of Biochemical Assays of the mutants
Table B1: Summary of SsoMCM Additional Mutations
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List of Figures

Fig 1: Overall features of the monomeric and hexameric ssoMCM structural
models
Fig 2: Structure based sequence alignment between ssoMCM and mtMCM
Fig 3: Comparisons of the three archaeal MCM structures
Fig 4: Structural features of the ssoMCM hexamer
Fig 5: Cis- and trans- interactions between the N- and C-domains of ssoMCM.
Fig 6: ATP binding interfaces
Fig 7: Structure-based mutagenesis and functional analysis of the mutants
Fig 8: Two possible DNA unwinding modes by MCM helicase
Fig 9: Electron density strength for ssoMCM
Fig 10: Overview of EXT-hp mutations
Fig 11: The results of FPLC and ATPase analysis
Fig 12: Results of DNA Binding Assays
Fig 13: The results of helicase assays
Fig 14: Possible DNA unwinding modes by MCM helicase.
Fig 15: The side channels at the C-domain and N-domains
Fig 16: Overview of LTag hexamer conformations and the β-hairpin structure
in different nucleotide binding states
Fig 17: The nucleotide pocket configurations in LTag and ssoMCM.
Fig 18: Nucleotide pocket closure in ssoMCM and the conformational changes
of the hexamer
Fig A1: Sequence alignment of Archaeal MCMs
Fig A2: Purifications of Archaeal MCMs
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Fig B1: Sequence alignment: ACL Loop
Fig B2: Sequence alignment, ATP pocket and side channel mutations
Fig B3: ATP Pockets of LTag and SsoMCM
Fig B4: Side channel and functional mutations
Fig C1: MCM3 homology model
Fig C2: MCM4 homology model
Fig C3: MCM5 homology model
Fig C4: MCM5 homology model
Fig C5: MCM6 homology model
Fig C6: MCM7 homology model
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Abstract
The mini-chromosome maintenance protein (MCM) complex is an essential
helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex
consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has
only one MCM protein (ssoMCM), six subunits of which form a homohexamer. Here, I
first discuss my 4.35Å crystal structure of the near full-length ssoMCM. The structure
shows an elongated fold, with five sub-domains that are organized into two large N- and
C-terminal domains. A near full-length ssoMCM hexamer generated based on the 6-fold
symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM)
hexamer shows inter-subunit distances suitable for bonding contacts, including the
interface around the ATP pocket. Four unusual -hairpins of each subunit are located
inside the central channel or around the side channels in the hexamer. Additionally, the
hexamer fits well into the double-hexamer EM map of mtMCM. Mutational analysis of
residues at the inter-subunits interface and around the side channels demonstrates their
critical roles for hexamerization and helicase function. I also present a series of 25
structure based mutations, 9 of 10 of which have been characterized by DNA binding,
ATPase and helicase assays. Finally, modeling of ATP binding and hydrolysis based on
the above evidence is presented.
1
Chapter 1: Introduction
Based on original introductions from the papers included in chapters 2 and 4
DNA replication in eukaryotes is a tightly regulated process involving many
different enzymes coming together to form complex machinery responsible for fast,
processive, and high-fidelity duplication of DNA, once and only once per cell cycle. The
active replication fork involves not only the DNA polymerase, but also single stranded
binding protein, primase, ligase, topoisomerase, helicase, the processivity clamp and a
variety of co-factors, all acting in a coordinated manner, and regulated to prevent both
early initiation and re-replication of DNA. Failure to regulate this system can lead to
aberrant cell division, or in the worst case, uncontrolled and rapid cell growth leading to
carcinomas (Forsburg 2004, Lau 2007).
In eukaryotes, Minichromosome Maintenance protein (MCM) is the DNA
helicase essential for unwinding genomic DNA during DNA replication (Forsburg 2004,
Moyer 2006, Bochman 2008). As a critical protein for cell division, MCM is also the
target of various checkpoint pathways, such as the S-phase entry and S-phase arrest
checkpoints (Forsburg 2008). Both the loading and activation of MCM helicase are
strictly regulated and are coupled to cell growth cycles.
Eukaryotic MCM consists of six gene products, MCM2-7, which form a
heterohexamer (Bell 2002). Each MCM gene has a series of conserved motifs, such as a
zinc-binding domain near the N-terminus of the enzyme and a helicase domain (called
the MCM box) with an ATPase core. However, the individual subunits differ. For
2
example, MCM2 and MCM3 have possible nuclear localization motifs but MCM4-7 do
not. MCM4 has a consensus sequence for phosphorylation by the cell cycle regulation
factor cyclin-dependant kinase (CDK) as part of replication initiation, while consensus
sequences among the other MCMs are more varied (Forsburg 2004).
The MCM box is a conserved motif comprising the majority of the C-terminal
region of all 6 MCMs. This region consists of an ATPase core with accessory hairpins
involved in helicase activity (see Chapter 2). The core falls into a diverse family of ATP
utilization enzymes known as AAA+ ATPases (ATPases associated with a variety of
cellular activities) (Iyer 2004). Consisting of a 5-strand β-sheet sandwiched between 5
alpha helices (3 “above” and 2 “below), the motif is well studied, with canonical residues
forming a pocket for ATP binding and hydrolysis. Most notable are the Walker A,
Walker B, sensors 1 and 2, and arginine finger residues. These residues are mostly
exposed at the ends of the strands of the β-sheet. The strands are numbered 51432, the
number referring to their occurrence in the primary amino acid sequence, and the order of
the numbers reflecting their layout in the sheet.
The ATP pocket is formed at a junction between two monomers. The “cis” side
of the pocket exposes residues that form a loop, called the phosphate binding loop (p-
loop) in which the tri-phosphate can bind. This loop, on strand 1, is formed by the
Walker A region, and includes a conserved lysine. The Walker B residues on strand 3,
typically DE, coordinate a water molecule used in nucleophilic attack on the ATP during
hydrolysis. The sensor 1 residue on strand 4 also assists in hydrolysis (Iyer 2004).
3
The “trans” monomer exposes a crucial arginine, the arginine finger, which
interacts with the ATP γ-phosphate. This inter-subunit binding facilitates conformational
changes communicated between subunits in the hexamer via binding and hydrolysis of
ATP (Iyer 2004). Finally, the sensor 2 residue is exposed on a separate α-helical bundle
which is found proximal to the ATPase core, but in different ATPases it can direct its
residue in cis- or in trans-. MCM has its senor-2 in trans- (Moreau 2007). Of interest,
the different MCMs do not have equal ATPase activity and appear to have differing
functions for unwinding DNA (Bochman 2009). This is discussed in more detail in
Chapter 4.
While for years the MCM 2-7 heterohexameric in vitro helicase activity could not
be demonstrated, recent reports have identified such activity, either in complex with the
replication factors cdc45 and GINS or separately (Moyer 2006, Bochman 2008). A great
deal of biochemical data has been collected regarding the heterohexamer, as well as the
dimer of the trimer MCM 4, 6, 7, which also exhibits helicase activity, unwinding DNA
with a 3’ to 5’ polarity (Ishimi 1997, You 1999, Lee 2001). However, high resolution
structural studies with heterohexameric eukaryotic proteins are lagging behind.
Archaeal replication systems share many similarities to eukaryotic systems,
including archaeal MCM proteins that are homologous to the eukaryotic MCM proteins
(Kelman 2005). Most archaeal genomes sequenced so far have revealed the presence of
only one MCM gene, whose products form homo-oligomers. This makes archaeal MCM
a simpler system for understanding MCM structure and function relationships. So far,
4
perhaps the best studied archaeal MCM proteins are from the species M.
thermoautotrophicum (mthMCM) and S. solfataricus (ssoMCM). Both proteins
oligomerize to form double-hexamers or hexamers in solution (Kelman 1999, Chong
2000, Carpentieri 2002, Fletcher 2003). Both exhibit single- and double-stranded DNA
binding, a basal ATPase activity stimulated by the addition of DNA, and 3’ 5’ helicase
activity on forked DNA substrate in vitro (Kelman 1999, Chong 2000, Carpentieri 2002,
Fletcher 2003, Pucci 2004).
In contrast to the lagging eukaryotic structural studies, recently there has been
great progress in the structural studies of archaeal MCM proteins, which has provided
valuable information for understanding MCM helicase mechanism. Currently, the
following X-ray structures have been determined: the N-terminal structures for mthMCM
(Fletcher 2003) and ssoMCM (Liu 2008), a near full-length ssoMCM structure
(presented in Chapter 2, from (Brewster 2008)), and an MCM homolog from another
archaeon, M. kandleri (mkaMCM2)((Bae 2009) and reviewed in (Sakakibara 2009)).
MkaMCM2 has a natural deletion of a critical zinc-binding subdomain in the N-terminal
domain, which prevents hexamerization and, thus, helicase activity. MkaMCM2 also has
non-canonical ATP-binding residues resulting in the elimination of ATPase activity (Bae
2009). Further, the most C-terminal ~70 residues that shares homology with a winged
helix fold is naturally deleted in mkaMCM2 (this domain was additionally not buildable
in ssoMCM, likely due to its flexibility).
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These new structures, together with the biochemical data presented in Chapter 3,
have allowed me to theorize regarding the MCM structure-function relationship. Many
questions are raised based on the data known so far, such as where is the DNA threaded
during unwinding? Does this threading relate to the many channels observed in the
structure? How does the oligomeric state of the protein relate to this threading? How
does ATP binding and hydrolysis effect conformational changes within the enzyme and
how is that coupled to helicase activity? What is the function of the hairpins in the
structure? In this thesis, I discuss the answers we have to these questions from the
structures and functional assays already performed, and then in Chapter 4 I speculate on
possible models of unwinding based on current biochemical and structural data.
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Chapter 2: Insights into Helicase Function from the Structure of a
Near-Full Length Archaeal MCM Helicase
Adapted from Brewster, A. S., G. Wang, et al. (2008). "Crystal structure of a near-full-
length archaeal MCM: functional insights for an AAA+ hexameric helicase." Proc Natl
Acad Sci U S A 105(51): 20191-6.
Structural features of the near full-length ssoMCM
We crystallized the full-length (FL, residues 1-686) and a C-terminal truncation
(T612, residues 1-612) of ssoMCM (Fig 1A, Fig 2). Se-SAD phasing was used to solve
the structures of the FL construct and the T612 construct. The molecular models built on
the electron density maps of the two constructs reveal a similar structure, both containing
the N-terminal domain and the C-terminal AAA+ domain, with one monomer per
asymmetric unit (Fig 1B). Even though low sigma level density for the winged-helix
domain (WH) at the most C-terminus is clearly present in the SAD map of the FL
structure, we could not build the WH domain due to the poorly defined electron density.
As a result, the final ssoMCM model contains residues 7-601, missing the C-terminal 85
residue WH domain.
The ssoMCM structure reveals an elongated fold, with well separated large N-
and C- terminal domains (Fig 1). The N-terminal domain consists of A, B and C
subdomains and appears to be used mainly for structural organization (Fletcher 2003, Liu
2008), and possibly for processivity (Barry 2007). Importantly, the N-domain is
strategically linked to the C-terminal AAA+ (ATPases Associated with diverse cellular
Activities) helicase domain through a long and conserved loop (Brewster 2008). This
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conserved loop, named the allosteric control loop (ACL), has been shown to play a role
in regulating interactions between the N- and C-terminal domains by facilitating
communication between the domains in response to ATP hydrolysis (Brewster 2008,
Sakakibara 2008, Barry 2009). Further, the N-terminal domain establishes the in vitro
3’ 5’ directionality of ssoMCM when added in trans- to the separated C-terminal
domain in vitro (Barry 2007).
A vital hairpin in the N-terminal domain (the NT-hp) projects into the central
channel where it binds DNA (Fletcher 2003, McGeoch 2005). Mutations involving
important basic residues on the tip of this hairpin weaken ssDNA and dsDNA binding,
and proportionally affect helicase activity in line with the lessening of DNA binding
(McGeoch 2005). The N-terminal -hairpin of N-ssoMCM is longer than that of the N-
mtMCM, due to a 5-residue insertion in the hairpin sequence of ssoMCM (Liu 2008).
Between the N- and C- domains is a 40-residue linker (N-C linker, in red in Fig
1A, B) that is well structured, forming a long connection with two consecutive helices at
its C-terminus that appear to be an integral part of the C-domain. The sequence of the C-
terminal domain is well conserved across species and between MCM subfamilies (Tye
2000). It consists of an AAA+ ATPase/helicase domain (the ATPase core), which is
composed of two clearly separable sub-domains: a canonical -helical/ -strand region
( / -domain, colored in cyan in Fig 1A, 1B), and a non-typical three -helical bundle
domain ( -domain, in purple in Fig 1A, 1B. See Supplementary Information for a more
detailed comparison with other known AAA+ protein structures). There are a total of 5
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main -strands and 5 main -helices in the / -domain, and 3 -helices in the -domain
(Fig 1B, and Fig 2). Connecting the / -domain and the -domain is a 47 residue linker
( / -  linker, in blue in Fig 1A, B). This / -  linker folds into two long -helices
spaced with a loop in the middle. Interestingly, the N-C linker and the distal   -  linker
wrap around each other like two interlocking index fingers (Fig 1B). Through such
Fig 1: Overall features of the monomeric and hexameric ssoMCM structural models

(A) A diagram depicting the domains of ssoMCM. N-C: N- to C- domain linker. / :
/  domain of ATPase core. / - : linker between sub-domains in the ATPase core. :
-domain. WH: winged helix domain (disordered in our structure). (B) Fold of ssoMCM
monomer. Domains and linkers are colored as in 1A. Helices are shown as cylinders and
-strands as arrows. Zinc atoms are shown as red spheres. -hairpins are labeled as
follows. NT-hp: N-terminal hairpin. H2I-hp: helix-2 insert hairpin. PS1-hp: pre-sensor 1
hairpin. EXT-hp: external hairpin. (C) Ribbon diagram showing the top and side views of
a hexamer model of ssoMCM.
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Fig 2: Structure based sequence alignment between ssoMCM and mtMCM

N-terminal ssoMCM/mtMCM sequence alignment adapted from (Liu 2008). ClustalX
was used to align the C-terminal portion (Thompson 1997). Secondary structure elements
for ssoMCM are shown on the top, with cylinders for helices, arrows for -strands, and
gaps for loops. The ssoMCM WH domain was not built in the structure and its secondary
structure is not listed. The N-terminal mtMCM secondary structures are shown below the
alignment. Helix and strand numbering are adapted from (Fletcher 2003, Iyer 2004, Liu
2008).

interlocking interactions, the two long linkers can stabilize not only the conformations of
each other, but also the relative positions of the N-domains and the C-terminal   - and
- domains.
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Other noticeable structural features in the C-domain include three -hairpin
structures protruding from the surface (labeled H2I-hp, PS1-hp, and EXT-hp in Fig 1B).
The most N-terminal -hairpin, named H2I-hairpin (or H2I-hp, resi 374-390), protrudes
farthest into the central channel of the hexamer model presented below, and has its -
hairpin tip structured into a small 3
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-like helix. This -hairpin is formed from a sequence
motif called “helix-2 insert” (H2I) that is unique to MCM and other H2I subfamily
members of the AAA+ ATPase family (Iyer 2004). Next to the H2I-hairpin is a shorter -
hairpin structure (resi 424-439}, which is formed from the sequence just ahead of (or N-
terminal to) the sensor-1 asparagine (Iyer 2004). As a result, this -hairpin is named the
pre-sensor-1 -hairpin (PS1-hp in Fig 1B). Comparable in length to the H2I-hairpin, but
more recessed from the central channel, this PS1-hairpin is equivalent to the major -
hairpin structure of the AAA+ domain of SV40 Large T antigen (LTag) hexameric
helicase (Li 2003, Gai 2004). The third -hairpin (resi 319-333) contains a VLED
sequence motif similar to that of the acidic pin of RuvA helicase, used to separate the
DNA fork during branch migration (Ingleston 2000). This -hairpin is located on the
exterior of the hexamer model, and is thus named EXT-hairpin (or EXT-hp in Fig 1B).
The mutational data reported here as well as from previous reports (McGeoch 2005,
Jenkinson 2006) demonstrate the critical role of these three hairpins for the helicase
function.
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Hexamerization of SsoMCM
SsoMCM exists mostly in hexameric form in solution with medium salt
concentration (see below). The structures of the N-domains of ssoMCM and mtMCM
show a similar hexamerization interface (Fletcher 2003, Liu 2008). These two align well
with each other (Fig 3A). However, N-ssoMCM has a narrower central channel largely
due to a longer -hairpin finger extending into the channel (Fig 3B, C) (Liu 2008). Based
on these two hexamer structures, we generated hexamer models of ssoMCM by applying
the 6-fold symmetry of either the N-ssoMCM or the N-mtMCM structures. Significantly,
the N-mtMCM 6-fold symmetry (PDB ID 1LTL) immediately yielded a hexamer that has
reasonable bonding distances between neighbors not only for the N-domain, but also for
the majority of the C-terminal AAA+ domain (Fig 1C). No clashes between neighboring
monomers are present in the hexamer structure. However, the hexamer model generated
using the N-ssoMCM hexameric symmetry (PDB ID 2VL6) has some clashes at the C-
terminal domain. Thus, it seems that ssoMCM may have two or more conformations
differing slightly in the angles between N- and C- domains, with at least one of such
conformations (as the one reported here) that can assemble a hexamer following the
hexameric symmetry of the N-mtMCM.
The ssoMCM hexamer model has a short dumbbell appearance, with a large N-
domain ring and C-domain ring on both ends, and a “slim waist” around the middle
portion of the hexamer (Fig 1C, side view). This general shape has been confirmed by
electron-microscopy (EM) studies (Pape 2003, Gómez-Llorente 2005, Costa 2006). The
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hexamer is 103 Å in length along the hexameric axis and 138 Å in width. The hexamer
has a wide central channel (Fig 1C, top view), narrowing towards the N-terminal end.
The central channel of the modeled hexamer has dimensions that can accommodate
ssDNA or dsDNA (also see (Fletcher 2003)). Clear side channels (11Å opening, between
main chain atoms) are present, which are comparable to the side-channels dimension
observed in the SV40 large T hexamer (12Å opening, between main chains atoms) (Li
2003, Gai 2004). Side-channels are also visualized previously by electron microscopic
(EM) reconstruction of mtMCM (Gómez-Llorente 2005, Costa 2006) and eukaryotes
(Remus 2009). These channels were proposed to be potential exits for unwound ssDNA
in the LTag double hexameric “looping” model of DNA unwinding (see discussion) (Li
2003, Gai 2004).
Fitting the hexamer into the mtMCM EM map
While ssoMCM has mainly been characterized as a hexamer, there is some
evidence it may form a double hexamer (Barry 2007). Double hexamer structures are
observed for its homolog mtMCM, with two hexamers stacked in a “head to head”
configuration (Fletcher 2003). We fitted the ssoMCM hexamer model to the available
EM map of the double hexameric mtMCM (Gómez-Llorente 2005). The overall topology
of the ssoMCM hexamer model fits snugly into the EM map (Fig 4A). Many major
structural features agree between the ssoMCM hexamer structure model and the EM map,
including the striking slim waist formed between the N- and C- terminal domains, the
side-channels, and even the surface contour inside the central channel.
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Fig 3: Comparisons of the three archaeal MCM structures: N-mtMCM (Fletcher 2003),
N-ssoMCM (Liu 2008), and the near full length ssoMCM (this report).
In all panels, mtMCM is in blue, N-ssoMCM is in magenta, and ssoMCM from this
work is in green. (A) Superimposition of N-mtMCM and N-ssoMCM hexamers.
Alignment by PyMOL (DeLano 2002). (B) Surface representation of N-mtMCM, N-
ssoMCM and ssoMCM, showing the differences in the central channel dimensions.
Approximations of central channels are shown as cylinders. Note, since ssoMCM is a
poly-alanine structure, we used the N-ssoMCM structure with side chains (Liu 2008)
to model the surface of the N-terminal domain, and programmatically mutated the C-
terminal side chains to their full side-chain sequence for making this panel. Narrowest
widths for the central channels are listed in angstroms, measured from side chain to
side chain. Poly-alanine models were also used to measure these distances, shown in
parenthesis. The C-terminal width of ssoMCM is from programmatically modeled
side-chains. (C) N-terminal domain of MCMs, viewed from the side to show the shift
of Zn domain (sub-domain B) and the shift of sub-domain A in the 3 structures.
Alignment done in PyMOL (DeLano 2002) and by Lovoalign (Martinez 2007) based
on sub-domain C from the hexamer models. Only the N-domain is displayed from
the near full length ssoMCM (this work). A, B, and C sub-domains are labeled.

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Fig 3, continued

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Fig 4: Structural features of the ssoMCM hexamer

(A) The double hexameric EM map of mtMCM with the ssoMCM hexamer model fitting
snugly inside the map (Gómez-Llorente 2005). The PS1-hairpin is located near the side-
channels of the EM map (indicated by an arrow). (B) Side and top views of the ssoMCM
hexamer model. Subunits are labeled a-f. Two subunits in the front are removed in the
side view to reveal the interior. The four -hairpins located inside the central and side-
channels are colored. (C) A close-up view of subunits a and b in the back side of the
hexamer in panel-B (side view). -hairpins are labeled as in Fig 1B. The opening
between the two neighboring subunits at the C-terminus (side-channel) is indicated. (D)
A close-up top view as in panel B, showing the radial and helical nature of the 4 β-
hairpins.

Structural features within the main channel
The narrowest point of the hexameric central channel is formed by the six NT-hairpins
within the N-terminal domains (Fig 4B-D). The next narrowest point is in the C-terminal
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helicase domain, which is formed by the H2I-hairpin (Fig 4B-D, Fig 3B). This hairpin
was previously predicted to be located in the central channel (Costa 2006, Jenkinson
2006), and the position may be relevant to its critical role in helicase activity (Jenkinson
2006). Specifically deletion of residues within the H2I-hp greatly enhances ssDNA and
dsDNA binding, but eliminates helicase activity (Jenkinson 2006). Further, the ATPase
activity of this mutant is not affected by the absence or presence of ssDNA, but was
stimulated 12-fold by the addition of dsDNA. These data led to the proposition that this
hairpin acts as a “plowshare” to separate the two DNA strands during unwinding (see
Chapter 4) (Jenkinson 2006).
In contrast to the H2I-hairpins that protrude into the central channel, the six PS1-
hairpins are somewhat recessed from the central channel (Fig 4B-D), as anticipated
before (Costa 2006). This is reminiscent of RuvB’s PS1-hairpin, which is recessed from
the central channel and forms contacts to RuvA (Yamada 2002). Unlike RuvB’s PS1-
hairpin, which likely does not interact with DNA (Ohnishi 2005), MCM’s PS1-hairpin is
involved in DNA binding and helicase activity (McGeoch 2005).
Again, the NT-hp is also involved in DNA binding but is not involved in helicase
activity. The double mutant with mutations on both the NT-hp and the PS1-hp lost DNA
binding completely (McGeoch 2005). Thus, these two hairpins are vital for interacting
with DNA in the channel, while the PS1-hp within the helicase domain has an additional
and more direct impact on helicase activity.
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The six PS1-hairpins of ssoMCM are also located near the C-terminal side
channel entrance that connects to the main channel. Such PS1-hairpin location makes it
accessible from both the main channel and the side channel (Fig 4B-D), and may have
functional implications for DNA unwinding.
Structure features around the side channels

In addition to the PS1-hairpin on the interior entrance of the side channel, the
acidic -hairpin (or EXT-hairpin) is present on the exterior exit of the side channel (Fig
4B-D). Unlike the PS1-hairpin that has positively charged residues on the hairpin tip, the
EXT-hairpin has two hydrophobic and two acidic residues (VLED324-327) on the tip. A
-hairpin with a similar motif in RuvA, called the acidic pin, is involved in DNA fork
unwinding during branch migration (Ingleston 2000). This acidic pin is situated on the
inside of the RuvA tetramer, as opposed to the exterior location of the EXT-hairpin in
MCM (see discussion below). SsoMCM D327 on the EXT-hairpin is well conserved
among archaeal MCMs and semi-conserved in eukaryotic MCMs (alignment not shown).
The presence of this acidic EXT-hairpin and the PS1-hairpin around the exit and the
entrance of the side-channel, suggests a potential role of the two -hairpins for helicase
function, potentially interacting with and translocating DNA through the side channel
(see discussion).
The residues from 199 to 211 form a well-structured loop (L207) in the N-
ssoMCM and N-mtMCM structures. This loop points towards the C-terminal domain. In
our ssoMCM hexamer model, it would abut against the PS1-hairpin and helix C 3 (Fig
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Fig 5: Cis- and trans- interactions between the N- and C-domains of ssoMCM
(A) Two monomers from the ssoMCM hexamer model are shown. Boxed regions
indicate the intra-subunit (or cis-) and the inter-subunit (or trans-) N-C domain
interactions shown in panels B and C. (B). A close-up view of the cis-contacts
between the N- and C domains, via the strand N 4 of the N-domain with the H2I-
hairpin of C-domain. This interaction appears to augment the -sheet of the N-
domain. Additionally, potential cis- interactions between L207 and the α/β domain
are shown. (C). A close-up view of the trans-contacts between L207 and an additional
loop that includes R110 of one subunit with the PS1-hairpin of the next subunit. In B
and C, residues mutated in mtMCM previously have been shown and colored
according to the effect on activity. R110A (orange, mtMCM R98) reduces DNA
stimulated ATPase activity of the C-domain. E199R and P210G (red, mtMCM Q181
and E182)) abrogated ATPase activity and helicase activity. Q198A, P201A, G207A
(green, mtMCM L184, E185, G190) increased ATPase activity. E202R (purple,
mtMCM P193) increased ATPase activity, WT helicase activity on forked substrates,
but abrogated helicase activity of “flat” substrates (3’ overhang only) (Jenkinson
2006, Sakakibara 2008). In B and C, black dashes indicate main-chain to main-chain
distances in angstroms.

19
Fig 5, continued

2) from a neighboring subunit around the side channel (Fig 5C). Mutational analysis of
this loop has been recently performed (Sakakibara 2008). This loop was proposed to act
as a medium for transmitting signals from ATP hydrolysis to the N-terminal domain. Our
hexamer structure indicates that these residues are within bonding distance with the α/β
domain of the same subunit (in cis-) and the PS1-hairpin/Cα3 region of a neighboring
subunit (in trans-) (Fig 5).
20
Nucleotide binding pocket at the interface

The ATP binding pocket, with its Walker A and B motifs on the ATP-bound
subunit and the arginine finger from the next subunit (in trans-), lies at the interface
between two neighboring subunits near the C-terminal end of the ssoMCM hexamer
model. There is no nucleotide in the crystal structure, and the ATP binding pocket
configuration of the ssoMCM hexamer model is similar to that of the empty site of the
LTag hexamer structure (Fig 6) (Gai 2004). The close resemblance of the ATP pocket
configuration to that of the LTag apo form provides further evidence that supports the
hexamer model presented here.
Like LTag helicase, the ATP binding pocket is C-terminal to the side channel.
The ring formed by the C-terminal domains of the hexamer model appears loose, a
characteristic of the apo LTag hexamer. The LTag hexamer tightens up upon ATP
binding to the ATP pocket at the interface between subunits (Gai 2004); the conformation
of ssoMCM hexamer model suggests that ATP binding should tighten the interactions
between two adjacent subunits in a ssoMCM hexamer.
Structure-guided mutational analysis of ssoMCM
We constructed six mutants of ssoMCM (M1-M6) based on the crystal structure
of ssoMCM in order to validate the structural model of the hexamer (Table 1, Fig 7). The
locations of the mutated residues on the structure are shown in Fig 7A. Among the six
mutants, the residues mutated in M1-M4 are located at the interface between neighboring
subunits in the hexameric model.
21
Fig 6: ATP binding interfaces
(A) Side view showing two monomers of the hexamer model of ssoMCM, with the
boxed region showing the location of the ATP binding pocket between two subunits.
(B) A close-up view of around the ATP binding pocket between two subunits from
the boxed region in panel A. The “ghost” ATP position is modeled based on LTag
ATP-bound structure (Gai 2004). ATP in gray to indicate it was modeled into the cis-
monomer (in light blue). Walker A (red), Walker B (orange) and sensor 1 (yellow)
residues are shown. From the trans- monomer (green), only the arginine finger is
shown (blue). (C) Electron density from our B-factor sharpened data showing correct
registry of the Walker A lysine in the poly-alanine structure. P-loop: phosphate-
binding loop. (D, E) ATP binding pocket from SV40 Large T antigen in the ATP
bound (panel D) and the apo- (panel E) state (Gai 2004), for comparison to the ATP
pocket configuration of ssoMCM in panel B. Panel E shows the withdrawn arginine
finger in the LTag apo- form, comparable to our apo- hexamer model.

22
Fig 6, continued

23
Table 1: Summary of ssoMCM mutational studies
# Mutation Location 250mM 1M Helicase
WT Hex Mon +
M1 L189D, D191R Interface (N-term) Mon* Mon - -
M2 A416R, A420R Interface (side channel) Mon* Mon - -
M3 TPDSP550GGGGG Interface (α-domain) Mon* Mon - -
M4 ILI555DSD Interface (α-domain) Mon Mon - -
M5 EEV202GGG Loop 207 (side channel) Hex Mon - -
M6 ED326AA, R329A EXT-hairpin Hex Mon -

The poly-Ala model was converted to a model with full side chains to aid mutational
design. For mutants with 3 or more residues mutated, such as M3 that replaces residues
550–554 with glycine, only the 1st mutated residue is numbered. WT, wild type; Hex,
hexamer; Mon, monomer; Mon*, predominantly monomer with a small hexameric
component; +,WT activity; -, significantly reduced activity; - -, near abrogation of
activity. M1–M4 are mutated residues at the intersubunit interface, and M5 and M6 are
functional mutants.

Fig 7: Structure-based mutagenesis and functional analysis of the mutants (also see
Tables 1, 2)

(A) Location of mutations on the ssoMCM monomer structure. (B) Superose-6 size
exclusion FPLC analysis of ssoMCM mutants in 0.25 M (blue line) and 1.0 M (pink line)
NaCl. The molecular marker positions are indicated. The calculated Mwt of ssoMCM
monomer is 77kD, hexamer 462kD. (C) Representative helicase assay. B: boiled dsDNA.
UB: un-boiled dsDNA. =: dsDNA. -: ssDNA. (D) Quantitative analysis of the helicase
assay of the mutants, shown as the percentage of WT activity. Error bars represent the
standard error from three experiments.
24
Purified wild type (WT) ssoMCM shows elution peaks consistent with the
molecular weight (Mwt) of a hexamer by gel filtration chromatography in a buffer
containing 0.25 M NaCl (Fig 7B), agreeing with previous reports (Carpentieri 2002,
Pucci 2004, McGeoch 2005, Barry 2007, Moreau 2007, Pucci 2007). Interestingly,
ssoMCM can shift to a smaller peak consistent with a monomer in 1.0 M NaCl. In
medium salt conditions (0.25M NaCl), where WT protein exists as hexamers, mutants
M1-M4 all eluted predominantly in the monomer peak, providing strong evidence
supporting the critical role of these residues in inter-subunit interactions for
hexamerization. Helicase assays revealed that M1-M4 mutants had essentially
undetectable unwinding activity (Fig 7C, 7D), suggesting these residues are important not
only for hexamerization, but also for helicase activity. Mutant M5 mutated residues on
the 3
10
-like helix in the N-domain L207 near the side channel (Fig 5C). Mutant M6
examines the functional role of the two acidic residues (ED) and the arginine on the tip of
the acidic EXT-hairpin located at the exit of the side channel. Both M5 and M6 formed
hexamers comparable to WT in the two salt concentrations (Fig 7B), suggesting they
maintained structural integrity. However, the helicase activity of both mutants was
greatly reduced (Fig 7C, 7D). This result suggests a critical role of both L207 and the
acidic EXT-hairpin around the side channel for helicase function.
To further examine the roles these mutations play in helicase function, we
performed DNA binding assays using fluorescence polarization anisotropy and ATPase
assays using the Enzchek phosphate release assay system (Table 2). Both ssDNA and Y-
shaped DNA were used for the binding assays. Among all six mutants, only mutant M4
25
had greatly compromised DNA binding and ATPase activities, which may correlate with
the fact that M4 is also the only mutant that has completely lost the hexamerization
ability under the tested conditions. In addition, M1 displayed very low level of binding to
Y-DNA, and M2 had greatly reduced ATPase activity. Perhaps most interesting mutants
are M3 and M5 that showed DNA binding and ATPase activity that are comparable to
those of the WT. Thus, the loss of helicase activity of these two mutants are not likely the
result of the change of properties in DNA binding and ATP hydrolysis, and mutations in
the two mutants somehow decoupled the DNA binding and ATP hydrolysis from the
strand separation of the dsDNA substrate.
Table 2: Kinetic parameters of ssoMCM mutants

ATPase activity ATPase activity + Y-DNA ssDNA binding Y-DNA binding
# k cat (min
-1
) K m (nM) k cat (min
-1
) K m (nM) K d (nM) K d (nM) Hill
WT 3.1±0.2 280±50 4.1±1.2 1900±1200 800±200 1000±100 2.5±0.7
M1 1.8±0.4 1200±700 5.4±0.9 13000±2000 2900±1400 Large K d
M2 Large K m Large K m 1200±500 4200±1900 1.5±0.5
M3 3.1±0.5 400±200 2.8±1.3 2500±2400 170±10 1200±200 2.1±0.6
M4 Large K m No detectable activity Large K d Large K d
M5 2.8±0.5 220±100 3.3±0.7 1600±800 200±70 1100±300 1.3±0.4
M6 2.7±0.7 4400±1900 Large K m 900±300 1400±200 2.1±0.6

Note:
1.
Large K
m
: K
cat
not informative under the conditions tested due to a high
K
m
value for the mutant, though some activity was detected.
2.
Large K
d
: > 20 µM.
Values are per monomer. Standard error is given for each value.

Discussion
The crystal structure of ssoMCM reveals the multi-domain organization of the
molecule as well as several unique structural features, including: four -hairpins, two
long interlocking inter-domain linkers, and the direct contacts between N- and C-
domains, both within a subunit (intra-subunit) and between subunits (inter-subunit) in a
26
hexamer model. A hexamer generated based on the 6-fold symmetry of the N-mtMCM
(Fletcher 2003) reveals inter-subunit distances suitable for bonding throughout the N- and
C-domains, including the conformations of the ATP binding pocket at the interface
between two subunits. Additionally, the hexameric model fits into the hexamer/double
hexamer 3-D EM map of FL mtMCM, and matches the surface contour and the striking
side channel openings of the EM map (Gómez-Llorente 2005). Furthermore, mutagenesis
of the residues at the inter-subunit interfaces and around the side channels shows the
critical role of these residues for hexamerization and for helicase function.

Mechanisms of N- and C-terminal communication
Previous structural and sequence analysis suggested a long linker bridging the N-
domain and the C-terminal helicase domain (Fletcher 2003). However, biochemical
evidence suggests that the N- domain of archaeal MCM communicates and interacts with
the C-terminal helicase domain (Jenkinson 2006, Barry 2007). This inter-domain
interaction is implied by the observation that the separately purified N-ssoMCM and the
C-ssoMCM cooperate in DNA binding and in helicase function (Barry 2007).
Additionally, an R98A mutation in the mtMCM N-domain (ssoMCM R110) reduces the
DNA-stimulated ATPase activity of the C-domain (Jenkinson 2006). Further, mutations
on L207 by us and others (Sakakibara 2008) were shown to affect ATPase and helicase
activity of ssoMCM and mtMCM. Our ssoMCM structure models reveal that the N- and
C-domains interact with each other, not only within a single subunit (cis- N-C
interactions, Fig 5B), but also between two subunits within a hexamer (trans- N-C
27
interactions, Fig 5C), despite a long 40 residue linker region between the N- and C-
domains.
Forming potential cis- N-C interactions within a subunit, the N-domain strand
N 4 is positioned next to the C-domain H2I-hairpin, such that the N-domain -sheet
appears to be expanded to the two strands of the H2I-hairpin (Fig 5B, see Fig 2 for helix
and strand naming). N-domain L207 is directed such that several of the previously tested
mutations could interact with the α/β domain of the same subunit. Possible trans- N-C
interactions in the hexamer model include L207 with both helix C 3 and the PS1-hairpin
from the C-domain of the next subunit (Fig 5C). Additionally, mtMCM R98 in the N-
domain, (R110 in ssoMCM), is located within bonding distance for interacting with the
PS1-hairpin of the C-domain from the next subunit (Fig 5C). These cis- and trans- N-C
interactions revealed by the hexameric MCM model provide a molecular explanation for
the observed co-operation of separately purified N- and C- domains, the reduced ATPase
activity of the mtMCM R98 N-terminal mutation, and the variety of L207 mutations on
the N-domain that affect ATPase activity and helicase activity (Jenkinson 2006, Barry
2007, Sakakibara 2008).
Location of C-terminal WH domain
The C-terminal WH domain was not modeled in our crystal structure. The poorly
defined electron density for this WH domain suggests a flexible domain position. Indeed,
evidence indicates different locations for the WH domain in the context of a hexamer.
FRET analysis suggests that the WH domain is on the side of ssoMCM hexamer and can
move toward the N-terminus to contact the N-domain (McGeoch 2005). This proposed
28
WH location would occupy the “valley” (or the waist) on the side of the hexamer (Fig
1C). However, the obvious “valley” is not occupied in the EM map of the double
hexameric mtMCM (Gómez-Llorente 2005, Costa 2006), suggesting a different location
for the WH domain. An alternative WH location is suggested from the EM study of the
DNA-bound mtMCM, which shows that the double hexamer has a C-terminal “cap”,
possibly consisting of the WH domain, at only one of the hexamers (Costa 2006).
Evidence suggests a third possible location for the WH domain. The EM maps of the
mtMCM double hexamer without DNA bound reveals weak density at the very C-
terminal end of the central channel (Gómez-Llorente 2005). If the WH were to be
modeled into our FL ssoMCM low-sigma level density map, it would also be located
around the very C-terminal end of the hexameric channel (not shown). Taken together,
these data suggest that the WH domain is likely a flexible appendage that adopts different
positions.
The multiple -hairpin structural elements in ssoMCM
One striking feature of the ssoMCM structure is that there are four -hairpin
structural elements located throughout the N- and C-domains (Fig 1B), with three
hairpins (NT-hairpin, H2I-hairpin, and PS1-hairpin) located in the main channel, and two
hairpins (PS1-hairpin and EXT-hairpin) located within the side channels (Fig 4B-D).
Interestingly, the three central channel -hairpins from one subunit are not arranged in a
straight line along the hexameric channel, rather, they are offset in such a way that the
NT-hairpin reaches over to the top of the H2I-hairpin of the next subunit, forming a
29
helical arrangement (Fig 4B-D), which may have implications in their interactions with
helical DNA substrates.
The PS1-hairpin is located at the intersection between the main and side channels.
In sequence alignments, this PS1-hairpin aligns with the lone -hairpin in the central
channel of SV40 LTag (Li 2003, Shen 2005). The LTag β-hairpin protrudes into the
hexameric central channel while the ssoMCM PS1-hp is recessed from the channel. We
have previously shown that the LTag -hairpin moves along the channel in response to
ATP binding and hydrolysis (Li 2003, Gai 2004), which is likely coupled to DNA
translocation and unwinding. Additional experiments will be required to determine
whether the PS1-hairpin, or other -hairpins of ssoMCM, will also have a similar
“power-stroke”, perhaps moving the hairpin towards the central channel, for DNA
translocation and unwinding. However, similar to the LTag -hairpin, the ssoMCM PS1-
and H2I-hairpins connect to the ATP-binding site through strands 2, 3 and 4 of the
AAA+ core, and border the ATP-site of the neighboring subunit. This structural
arrangement suggests that ATP binding/hydrolysis may also be able to trigger the
movement of the PS1-hairpin and the H2I-hairpin for helicase function. Indeed, mutation
of residues on the PS1-hairpin in ssoMCM, or deletion within the H2I-hairpin sequence
in mtMCM, abolishes helicase activity (McGeoch 2005, Jenkinson 2006), suggesting a
critical role in helicase function for these two -hairpins (Chong 2005, Takahashi 2005,
Jenkinson 2006).
The acidic -hairpin located at the exit of the side channels is also directly
connected to the ATP P-loop through -strand 1. Therefore, the EXT-hairpin may also
30
respond to ATP-binding/hydrolysis. Our mutational data demonstrated that the residues
at the tip of the EXT-hairpin are vital for helicase activity (mutant M6, Fig 7, Table 2)
but not DNA binding. Additionally, an arginine mutation at the base of the EXT-hairpin
(R331A) removes ATPase and helicase activity (Moreau 2007), which also suggests the
important role of the EXT-hairpin for function. The location of the EXT-hairpin at the
side channel exit, together with the PS1-hairpin at the side channel entrance from the
central channel, suggests an intriguing possibility that these two -hairpins may work
together to interact with DNA passing through the side channels during unwinding.

Potential DNA unwinding modes by ssoMCM
Based on the structural and biochemical data of ssoMCM, two possible DNA
unwinding modes for the hexameric ssoMCM are represented by simple cartoons in Fig
8. The locations of the four -hairpins of ssoMCM are schematically shown in a two-
dimensional hexamer diagram in Fig 8A. One unwinding model (Fig 8B) is similar to the
“wedge” (or steric exclusion) model proposed for DnaB (Lee 2001, Kaplan 2003,
Rothenberg 2007), with one DNA strand passing through the central channel, and the
other being excluded from the channel. The PS1-hairpin seems less accessible in this
unwinding mode, but the EXT-hairpin may directly participate in coordinating the
position of the 5’ strand, perhaps disengaging the 5’ strand during unwinding, providing a
basis for the “opposite strand interaction” model proposed recently (Rothenberg 2007).
31
The second unwinding model in Fig 8C shows a hexameric helicase binding a
dsDNA region ahead of the fork, extruding ssDNA strands from a side channel. In this
model, the three -hairpins in the helicase domain all interact with DNA directly during
unwinding, as does the NT-hairpin. The unwinding modes presented in both models in
Fig 8A and B can be adapted to suit a double hexamer helicase. The validation of these
models requires further studies.
In this chapter, the crystal structure of near FL ssoMCM is described, which
reveals several new structural features and uncovers the multi-domain organization of FL
MCM, both as an individual subunit, and in a hexameric model. Moreover, our structure

Fig 8: Two possible DNA unwinding modes by MCM helicase

(A) Schematic representation of a MCM hexamer helicase. The four -hairpins
(NT, H2I, PS1, and EXT-hairpins) are represented by short solid bars, the central channel
and the side-channels are in darker shades. (B) Steric exclusion model for a single-
hexameric MCM helicase. (C) Side-channel extrusion model, showing ssDNA extruding
from the side channel. DNA is shown as black lines. Arrows indicate direction of helicase
movement.
32

-based mutational data provide experimental evidence supporting the important role of
several key structural features, including that of the MCM hexamerization interface for
helicase function. These structural and biochemical data provide a foundation for future
investigation of the functional role of archaeal and eukaryotic MCM complexes in DNA
replication.
Methods and Materials
Crystallization, data collection and structural determination
The FL MCM construct (residues 1-686) and a truncation mutant T612 (residues
1-612) have been crystallized (see Supplementary Information for details), and native and
Se-Met diffraction data were collected (Table 3). Experimental phases to 4.6Å and
4.35Å resolution were determined for both constructs using SAD data. The phases were
further improved by density modification using solvent flattening and histogram
matching. The improved electron density maps from both FL and T612 are very similar
to each other, with the T612 map having more featured helices due to slightly higher
resolution. Secondary-structure elements and domain organization are clearly
recognizable in most parts of the density map, as expected for the resolution range of the
crystallographic map. The N-ssoMCM crystal structure (PDB ID 2VL6) was immediately
docked into the N-domain of the map by automated phased translation searches. The 
and  core domain ( / -domain) taken from the bchI and cdc6 AAA+ domain (PDB IDs
1G8P, 1FNN) (Liu 2000, Fodje 2001) was subsequently docked into the map
automatically using the phased translation searches and re-built. Other helices and loops
33
were built using the graphics program COOT. To the best of the author’s knowledge, the
amino acid registry is correct; however chain-trace registry errors may still be present due
to the poly-alanine nature of the structure. A more detailed description of structural
determination is included in the supplementary materials below and Fig 9.
Supporting Information
Cloning, protein purification and gel filtration chromatography
FL ssoMCM was cloned into the pGEX-KG to express as a GST fusion protein.
Truncations and mutations were confirmed by sequencing the entire ssoMCM coding
region. Recombinant proteins were purified from E. coli using glutathione resin in a
buffer containing 50mM Tris-Cl (pH 8.0), 250mM NaCl, 5 mM dithiothreitol (DTT).
Following Thrombin cleavage to remove the GST fusion, soluble MCM protein was
further purified by Superose-6 column chromatography and concentrated to ~30 mg/ml in
a buffer containing 25mM Tris (pH 8.0), 50mM NaCl, and 5mM DTT. For the
oligomerization assay, protein at a concentration of 5 mg/ml was first equilibrated into
50mM Tris-Cl (pH 8.0), 5 mM DTT and either 0.25 or 1.0 M NaCl, and then analyzed by
gel filtration chromatography on an analytical Superose 6 column at 4°C.
A Detailed Description of the Crystallization Conditions
The FL MCM construct (residues 1-686) crystallized in a buffer containing 0.1M
sodium acetate (pH 4.9), 250 mM NaCl, and 20% MPD using the hanging drop vapor
diffusion method. Crystals of the truncation mutant T612 (residues 1-612) grew in a
buffer containing 0.1 M sodium acetate (pH 4.6), 50mM NaCl, 65mM CalCl
2
, and 20%
34
MPD. While these salt conditions should support hexamerization in solution (see results),
likely the low pH or MPD conditions promoted disassociation. The space group for the
crystals of both the FL and the T612 is I4
1
22, with near identical cell dimensions. Many
techniques were attempted to improve diffraction, including additive screening,
dehydration, and cryoprotectant screening. The best diffraction data were collected to
4.35Å resolution from selenomethionine (Se-Met) and native crystals (Table 3).
A Detailed Description of the Structural Determination Of ssoMCM
To determine the ssoMCM structure, Se sites were located using Phenix.hyss
(Adams 2002) from the Se-SAD datasets for both the FL (eleven sites) and T612 (ten
sites) constructs. Phasing from these sites was performed by CNS to 4.35Å resolution for
T612 or 4.6Å resolution for FL construct crystals (Brunger 1998, Brunger 2007). The
electron density maps were further improved by density modification and cross-crystal
averaging using CCP4, also generating a native 4.35Å map (1994). The quality of the
map was sufficient for building the model as follows. The N-ssoMCM crystal structure
(PDB ID 2VL6) was first automatically docked into the map by phased translation
searches (Strokopytov 2005). The model was further adjusted manually in O and COOT
to fit some loops into the density (Jones 1991, Emsley 2004). For the remaining C-
terminal domain electron density, two related AAA+ domain structures of bchI and cdc6
could not be fitted into the map. However, the characteristic AAA+ / -domain taken out
of the bchI or cdc6 structures (PDB IDs 1G8P, 1FNN) (Liu 2000, Fodje 2001), which
contains five -helices around a 5-stranded -sheet core, could be docked nicely into the
electron density map at the C-domain by automated phased translation searches
35
(Strokopytov 2005). Again, manual adjustment of some regions was necessary to fit the
map.
After docking these two major domains into the map, electron density for seven
more -helices and loops located outside the two fitted domains was still unoccupied and
needed to be modeled. Three of the four -helices of the -domains taken from cdc6 and
bchI were then modeled into a region containing a three -helix bundle that corresponds
to the -domain of ssoMCM. The remaining -helices and the loops were built manually
to complete the model. At the resolution range of 4.35Å, with no NCS averaging
available, only a poly-alanine model could be built. The maps calculated with sharpened
data (DeLaBarre 2006), by applying a B factor of –150, were better featured than the
unsharpened data, and were used along side the map calculated using the unsharpened
data in the process of the model building. Only weak density corresponding to the WH
domain was visible at the very C-terminus of the FL construct. As a result, the WH
domain was not built into the final model.
Geometry restrained refinement of the poly-alanine model with REFMAC5
(Murshudov 1997) was performed using the Se-SAD data set between 30-4.35 Å
resolution, which yielded R
work
and R
free
of 42.0% and 47.9% respectively (Table 3).
Further refinement of the structure did not improve the R factors due to the resolution
limitation of the data. The structure has good geometry and Ramachandran plot statistics
(Table 3), which are comparable or better than some of the published structures at a
similar resolution range (Chen 2005, DeLaBarre 2006, Jenni 2006, Maier 2006, Lomakin
2007, Sirajuddin 2007). Even though the registry of the model may await further
36
verification with higher-resolution data, the structural model is supported by the
following evidence. First, after replacing the poly-alanine with the full amino acid side
chains of ssoMCM, most hydrophobic side chains are buried, and the hydrophilic side
chains are exposed. Second, the buried residues make reasonable hydrophobic packing
interactions. Third, the Se-Met side chains fit nine out of the ten Se sites well, providing
confidence for the model registry. The tenth Se atom found by Phenix.hyss did not align
to a methionine and its removal did not affect the quality of the electron density map,
suggesting that it may be a spurious site. Fourth, the p-loop residues for ATP binding
pocket, including the Walker A and Walker B motifs, are in the canonical positions ready
for interacting with ATP (Fig 6). Fifth, molecular replacement solutions can be found
using the structural model against different native data sets of FL or T612 constructs, and
good maps can be obtained using the resultant MR model and the corresponding data
sets, indicating calculated phases from the model were reasonable. Sixth, the structure-
guided mutagenesis results provide direct experimental evidence supporting the structure
model. Nonetheless, there are some regions with weak density, most of which correspond
to locations of loops. These parts of the model may be more prone to errors, and their
locations are shown in Fig 9.
37
Table 3: Crystallography Statistics

SeMet
1
Native SeMet
Full length T612 T612
Data collection
Space group I4122 I4122 I4122
Cell dimensions   
   a, b, c (Å)
203.445, 203.445,
127.238
203.112, 203.112,
129.286
202.526, 202.526,
128.243
           ( )  90, 90, 90 90, 90, 90 90, 90, 90
Wavelength 0.9791 Peak 1.0000 0.9794 Peak
Resolution (Å) 50 - 4.60 30 - 4.35 30 - 4.35
R sym (%)
2
9.5 (65.7) 6.0 (46.5) 7.8 (67.0)
I / I 33.8 (1.4) 35.9 (4.6) 49.3 (4.4)
Completeness (%) 98.3 (92.4) 99.8 (100.0) 99.9 (100)
Redundancy 12.8 (7.9) 6.1 (6.1) 12.4 (12.8)
No. observed reflections 99113 55298 112095
No. unique reflections 7760 9101 9058.0
Phasing
Phasing power 1.25 (.33) 1.40 (0.41)
R cullis (%) 66.8 (73.3) 66.1 (87.6)
Figure of merit 0.20 0.26
Figure of merit (post DM)
3
0.90 0.91
Refinement
R work / R free (%) 41.2/48.1
No. atoms (protein) 2975
R.m.s. deviations
Bond lengths (Å) 0.008
Bond angles ( ) 1.195
Residues in disallowed regions (%) 0.7
1
SeMet: selenomethionine derivative.
2
Number in parenthesis: value for the highest resolution
bin.
3
DM: density modification.

38
Fig 9: Electron density strength for ssoMCM

SsoMCM is colored according to electron density presence in the map. Green: residue
fully within density bounds. Yellow: residue at or near a density boundary. Red: very
weak or no density.

Fitting the structure model into the EM map
Fitting of the hexamer structure model into the EM map was done by Chimera
using the double hexameric mtMCM map reported previously (Gómez-Llorente 2005).
We first used two ssoMCM hexamers to create a double hexamer model based on the
matrix generated from double hexameric mtMCM (Fletcher 2003).
Experimental Procedures for helicase assays, DNA binding, and ATPase assays
For the helicase assay, a fork DNA or Y-DNA including 44nt single-stranded tails
and a 44nt duplex region was used as the substrate. This Y-DNA substrate was obtained
39
by annealing two complimentary strands followed by
32
P- labeling. Sequences of the
oligonucleotides are
(dT)
44
GCTCGTGCAGACGTCGAGGTGAGGACGAGCTCCTCGTGACCACG and
CGTGGTCACGAGGAGCTCGTCCTCACCTCGACGTCTGCACGAGC(dT)
44
.
Helicase assays were performed by incubating the dsDNA with wide type or mutant
ssoMCM at 65 °C for 60 min in a 20 L volume containing 0.5 nM dsDNA, 75nM (as
monomer) of the indicated protein, 5 mM ATP, 1 mM DTT and 0.1 mg/ml BSA in 1x
helicase buffer (30 mM Tris-Acetate (pH 8), 75 mM NaCl, 50 mM potassium acetate, 10
mM Magnesium acetate). The reaction was terminated by adding 5 L of stop solution
containing 100 mM EDTA, 0.5% SDS, 0.1% xylene cyanol, 0.1% bromophenol blue and
50% glycerol. Aliquots were then loaded on a 12% polyacrylamide gel in 1 
Tris/borate/EDTA buffer for 50 min at 150V. The unwinding of the substrate DNA was
detected by autoradiography and quantified.
DNA binding was measured using fluorescence polarization anisotropy in a
QuantaMaster QM-1 fluorometer (Photon Technology International). A 70 µL reaction
containing 50 nM ssDNA or Y-DNA labeled with 6-FAM at the end, in helicase buffer
was titrated with 0.25-8.5 µM (as monomer) protein at 25°C. Anisotropy was measured
and K
d
was calculated as described previously (Greenleaf 2008). Experiments were
performed in triplicate.
ATP hydrolysis and phosphate release by ssoMCM was measured using the
Enzchek phosphate release assay kit (Invitrogen). All reactions were carried out in
helicase buffer at 65 C, using 1 µM (as monomer) protein and 50-2500 µM ATP.
40
Stimulation by Y- DNA was measured in the presence of 300 nM fork DNA (described
above). Data was fit to the Michaelis-Menten equation. Experiments were performed in
duplicate.
MCM helicase domain structure compared with known AAA+ structures
It is interesting to observe that the 3 helix α-domain in ssoMCM is relocated from
its typical position in AAA+ ATPases to the opposite side of the 5-strand α/β-α domain
by a long linker (α/β-α linker, blue in Fig 1). While this may be an artifact from the
crystal structure, its location has been predicted previously (Fodje 2001, Iyer 2004,
Erzberger 2006). The long linker, including helices Cα5 and Lα3 (see Fig 2 for helix and
strand naming), interlocks with the long N-C linker with its two helices Lα1 and Lα2.
Helix Lα3, along with its connecting loops, comprises the “pre-sensor II insertion”
described previously that defines the “PS-II” sub-clade within the PS1 family (Erzberger
2006). This arrangement moves the sensor-II arginine from its classical cis- position to
act in trans-, as biochemical evidence has shown (Hansson 2002, Erzberger 2006,
Moreau 2007).
In another AAA+ protein, bchI, a magnesium chelatase also in the PS-II clade, it
is argued that helices Cα6 and Cα7 from the α domain abut helix Cα5 in trans-,
reconstituting the canonical 4 helix α-domain (Erzberger 2006). This is not observed in
our hexamer model, though the α-domain is in close proximity to the Cα5 of the next
monomer; ATP binding could bring the helices together, assuming the Cα5 interaction is
conserved in MCM. Regardless, mutations M3 and M4 from this work, in the unordered
loop connecting helices Cα6 and Cα7, affect hexamerization and abolish helicase
41
activity. M4 further greatly compromises ATPase activity and DNA binding. This loop
could be directed towards helix Cα5. It is tempting to speculate that it could coordinate
helix Cα5 during hexamerization, but it should be noted that bchI does not contain a long
α-domain loop as does ssoMCM.
It has been suggested based on biochemical data that the -domain of bchI
relocates in response to ATP binding back to the typical α-domain position (Hansson
2002, Iyer 2004). Others argue that many hydrophobic contacts along the α/β-α linker
and α-domain with the α/β core, along with contacts in trans- between the α domain and
Cα5, would make this difficult (Erzberger 2006). Our structure of ssoMCM further shows
that it would be unlikely for the -domain to relocate to a different position, as its /  α
linker is “pinned down” and interlocked with the N/C linker (Fig 1B). If relocation upon
ATP binding of the α-domain is a common feature of PS-II family members, MCM may
prevent relocation via this pinned structure, re-channeling the motion for other purposes.
Alternatively, ATP binding could somehow relieve the interlock between the two linkers,
which could then allow the -domain to relocate.

The PDB has been deposited in the RSCB as PDB ID 3F9V.
42
Chapter 3: Mutational Analysis of an Archaeal MCM Exterior Hairpin
Reveals Critical Residues for Helicase Activity
Adapted from a manuscript currently in submission.

One of the major structural features of the subunit structure of ssoMCM is the
four obvious -hairpins projecting from the monomeric ssoMCM. One, located in the N-
terminal domain (NT-hairpin), projects into the central channel and has been implicated
in DNA binding (Fletcher 2003, McGeoch 2005). The other three -hairpins are located
in the C-terminal AAA+ domain: the pre-sensor 1 hairpin (PS1-hp), the helix 2 insertion
hairpin (H2I-hp), and the external hairpin (EXT-hp) (Brewster 2008).
Residues on the PS1-hp and H2I-hp play a role for helicase activity, are involved
in DNA binding and project into or near the central channel (McGeoch 2005, Jenkinson
2006). The EXT-hp, however, is located on the exterior side of the hexamer, near the
side channels in the C-terminal domain (Brewster 2008). The unusual location of this
EXT-hp raises interesting questions regarding its functional role. However, unlike the
other -hairpins, no detailed mutational and functional analysis of the EXT-hp has been
performed, and its role in DNA binding, ATPase, and helicase activity is not understood.
In this work we present a granular examination of this important structural feature,
including a through examination of the hairpin’s role in DNA binding, ATP hydrolysis
and helicase activity.
Experimental Procedures
Cloning, Purification, and Size Exclusion Analysis
43
WT Sulfolobus solfataricus MCM and mutations were cloned and purified in
wash buffer (WB: 50 mM Tris-Cl (pH 8.0), 250mM NaCl, 1 mM DTT), as described
previously (Brewster 2008) with the following changes: prior to size-exclusion
chromatography, the protein was first purified on a 6 mL Resource Q anion exchange
column. Further, after purification, the protein concentration was assayed by nano-drop
and SDS-PAGE analysis. The protein was then diluted to 10 uM in helicase buffer (HB,
30 mM Tris acetate (pH 8), 75 mM NaCl, 50 mM potassium acetate, 10 mM magnesium
acetate), aliquoted, and frozen at -80ºC. ~0.5 mg of protein was taken separately to
analyze on size-exclusion chromatography.
Helicase Assays
Helicase assays were performed exactly as described previously on radio-labeled
Y-shaped DNA substrate (Brewster 2008). The substrate was created from annealing two
complementary strands. The sequences are: (dT)
44
GCTCGTGCAGACGTCGAGGTGAGGACGAGCTCCTCGTGACCACG (strand Y1)
and CGTGGTCACGAGGAGCTCGTCCTCACCTCGACGTCTGCACGAGC (dT)
44

(strand Y2). Experiments were performed in triplicate.
Electrophoretic Mobility Shift Assays
DNA binding constants were determined using electrophoretic mobility shift
assays (EMSAs). ssDNA (strand Y2) or Y-shaped DNA from above was radio labeled
and desalted using a Micro Bio-Spin 6 Column (BIO-RAD) . Increasing amounts of
protein were incubated with 1.4 nM DNA in DNA binding buffer (DB, 20 mM Tris-Cl
(pH 7.5), 100mM NaCl, 2mM EDTA, 0.5 mM magnesium chloride) for 30 minutes at
44
65ºC, then ran on a 5% polyacrylamide gel in 0.5x Tris/borate/EDTA buffer for 90
minutes at 100 V. DNA bands were detected by autoradiography and quantified. %
DNA bound was determined vs. protein concentration and K
d
was calculated as described
in (Greenleaf 2008). Experiments were performed in duplicate.
ATPase Assays
ATPase assays were performed as described previously (Brewster 2008).
Experiments were performed in triplicate.
Results
Mutational Analysis of the exterior hairpin
The EXT-hp sequence is semi-conserved in archaea and eukaryotes (Fig 10A),
which raises intriguing questions as to the functional role of this hairpin next to the side
channel on the exterior of the hexamer. Previously, we showed an EXT-hp triple mutant
was deficient in helicase and ATPase activity (Brewster 2008). To further explore this
hairpin’s role in helicase function, we created a series of 10 single and double alanine
mutants (numbered M1 to M10), 9 on the EXT-hp and 1 control mutation of the Walker
A lysine involved in ATP binding and hydrolysis (Fig 10B). The 9 hairpin mutants are
located in three regions, an inside β-strand that faces towards the interior side of the
hexamer and side channel, a tip, and an outside β-strand that faces away from hexamer
(Fig 10C, 10D).

45
Fig 10: Overview of EXT-hp mutations
A) Sequence alignments of MCMs across archaea (top) and eukaryotes (bottom), for the
short region corresponding to the EXT-hp. Sso: Sulfolobus solfataricus. Sac: Sulfolobus
acidocaldarius. Prn: Aeropyrum pernix. Mth: Methanothermobacter
thermautotrophicus. Pyf: Pyrococcus furiosus. Tap: Thermoplasma acidophilum. Sp:
Schizosaccharomyces pombe. Sc: Saccharomyces cerevisiae. Hs: Homo sapiens. Xl:
Xenopus laevis. Dm: Drosophila melanogaster. At: Arabidopsis thaliana. B) Table of
mutations used for this work. C) Tilted side view of the ssoMCM hexamer model,
showing the EXT-hp on the external side of the hexamer and near the side channel. A
single subunit is shown in cyan, with its EXT-hp in red. The ATP binding pocket and
side-channel are indicated. D) Close up view of EXT-hp. Inside and outside refer to
towards the central channel and away from the central channel, respectively. The
locations of the residues mutated in this study are colored on the EXT-hp according to
amino acid type, as in panel B.
46
Fig 10, continued

Mutant M1 changes a highly conserved lysine on the exterior side of the hairpin,
but close to the hairpin base. Mutants M2-M7 bear single or double mutations on the
VLED sequence that wraps around the tip. We previously postulated that these residues
could function as a repellent to DNA, similar to the acidic pin in RuvA. M2-M4 change
the properties of the hydrophobic residues, VL. Of the two, the leucine is more
47
conserved but neither are strongly conserved in eukaryotes or archaea. M5-M8 mutate
the acidic ED region. Of note, the aspartate is highly conserved in archaea, but in
eukaryotes its conservation varies by homolog. For example, in MCM5 it remains an
aspartate, but in MCM3 it is consistently changed to asparagine. M8 changes a well
conserved arginine in archaea that only remains consistently basic in MCM 7, and mostly
so in MCM 2. M9, changing an absolutely conserved arginine at the base of the hairpin,
had been assayed previously for ATPase and helicase activity (Moreau 2007), and was
included as an additional control. We further assayed it for DNA binding differentials.
Finally, M10, the Walker A mutant, changes the conserved lysine vital for ATP
hydrolysis (Chong 2000). The Walker A motif is not conserved in the inactive MCM
homolog from Methanopyrus kandleri (Bae 2009), nor is the EXT-hp present in that
isoform.
Oligomerization as assayed by size-exclusion chromatography
We first assayed oligomeric state to determine if these mutations would affect
folding or hexamerization as compared with the wild type. At 250 mM NaCl, wild type
ssoMCM elutes on a Superose 6 size exclusion column as a hexamer. We found
previously that mutations that affect hexamerization elute as two peaks, with one at lower
oligomeric state, or shifting entirely to a lower oligomeric species (Brewster 2008). As
shown in Fig 11A, all the 10 mutants reported here eluted predominantly as a hexamer at
250 mM NaCl, indicating that none of the mutants has obvious defects in
oligomerization.
48
Fig 11: The results of FPLC and ATPase analysis

A) FPLC analysis of the mutations by gel filtration chromatography on a Superose-6
column. Molecular marker positions, Ferritin (440 kD) and aldolase (158 kD), are
indicated by arrows). B) SDS-PAGE gel analysis of the purified mutant proteins. C)
ATPase activity curves for WT, M3 and M8 in the presence and absence of Y-shaped
DNA. Error bars representing standard error of the mean are present, but in most cases
are too small to see. D) Summary of ATPase data in the absence (black bar) and
presence (white bar) of Y-shaped DNA. Error bars are standard error from curve fitting.
49
ATPase activity
We next performed a series of biochemical assays of the mutants to examine their
effect on ATP hydrolysis and other activities. Protein concentrations for these assays
were calibrated by nano-drop and further confirmed by SDS-PAGE in Fig 11B. The first
set of assays determined k
cat
and K
m
values for ATP hydrolysis via the Enzchek
phosphate release assay, as described previous (Brewster 2008). Activity curves for
wild-type and mutants M3 and M8 are shown in Fig 11C. For each experiment, an
additional test was performed to measure ATPase stimulation upon addition of Y-shaped
fork DNA. WT shows a modest stimulation by the Y-shaped DNA, as noted previously.
M3’s ATPase activity is significantly higher than WT’s. M8’s ATPase activity is lower,
and furthers loses ATPase stimulation upon the addition of DNA. A summary of k
cat

changes, together with the full data, including K
m
values, is given in Fig 11D and listed in
Table 4.
DNA-binding activity
DNA-binding activity of the mutants was determined by EMSAs using a single stranded
and forked DNA substrate (Fig 12, binding constants in Table 4). Generally speaking,
mutations of the hydrophobic tip (mutants M2-M4) increased DNA binding slightly.
Mutations in the conserved aspartate on the tip of the hairpin (M6) showed a large
decrease in dsDNA binding, and the double acidic mutant (M7) had a similar decrease in
ssDNA and dsDNA binding. All of the basic mutations, including M1 on the outside of
the hairpin, decreased ssDNA binding somewhat, and dsDNA binding significantly, with
M8, R329A having the largest effect. These findings indicate that this
50
Table 4: Kinetic Parameters of EXT-hp mutants
ATPase activity ATPase + Y-DNA ssDNA Binding Y-DNA Binding
Mutant k
cat
, min
-1
k
m
, nM k
cat
, min
-1
k
m
, nM K
d
, nM Hill K
d
, nM Hill
WT 3.1±0.1 170±20 3.8±0.1 190±20 240±0.5 8.8±0.5 224±7.3 5.8±1.3
M1 1.8±0.1 140±30 3.7±0.5 440±150 317±5.8 4.4±0.3 398±4.0 13.2±1.3
M2 2.4±0.1 120±20 3.6±0.1 160±20 209±5.8 4.7±0.5 230±7.2 10.1±3.3
M3 4.7±0.3 270±40 5.2±0.2 260±30 169±1.3 5.6±0.1 186±6.4 7.9±1.1
M4 3.9±0.1 230±10 5.3±0.2 260±30 165±10.0 13.4±4.3 178±1.5 7.7±0.5
M5 2.9±0.2 60±40 3.7±0.2 180±30 185±0.3 12.1±0.3 224±0.9 11.6±0.4
M6 2.3±0.1 100±20 2.6±0.2 130±30 283±78.7 15.6±20.1 400±6.5 5.6±0.4
M7 2.0±0.1 90±10 2.2±0.1 90±20 444±1.0 10.6±0.5 404±2.6 8.8±0.4
M8 1.8±0.1 170±40 1.7±0.1 170±30 380±1.8 8.0±0.3 483±0.4 9.0±0.03
M9 No Activity No Activity 325±8.3 6.2±0.8 428±2.0 12.9±0.5
M10 No Activity No Activity 267±7.0 4.5±0.5 339±2.6 11.4±2.0

All values are calculated based on monomeric MCM subunit. For each value, standard
error is given. Hill: Hill cooperativity coefficient.

hairpin is involved in DNA binding. Additionally, we see highly cooperative DNA
binding in our assays. Previously, using flourescence polarization anisoptropy, we had
seen Hill factors for Y-DNA binding ranging from 1.3 to 2.5 (Brewster 2008), suggesting
cooperativity of DNA binding. Now, using EMSA assays, we see Hill factors ranging
from 4 to as high as 15 for both ssDNA and Y-DNA, confirming the nature of
cooperativity of DNA binding, which is likely related to the oligomerization of six
subunits of ssoMCM upon DNA binding. The higher Hill factors reported here are likely
from the differences in experimental conditions; for example we have added a heating
step by treating the protein-DNA mixture at 65ºC for 30 minutes during sample
preparation, which may allow the protein to bind DNA better.
51
Helicase activity
Helicase assays were then performed on these mutants using radio-labeled forked
DNA substrates (Fig 13). As expected, the R331A and Walker A mutations, which had
no detectable ATPase activity, exhibited no helicase activity, as shown previously
(Chong 2000, Moreau 2007). Generally speaking, those mutants with lower ATPase and
DNA binding activity also has lower helicase activity. However, the deficits in ATPase
and DNA binding of some of the mutations did not strictly correlate with the level of
deficits in helicase activity. In particular, the acidic mutations that showed significant
decreases in DNA binding (M6 and M7), and decreases in ATPase activity, did not suffer
significant decreases in helicase activity, at least under the assay conditions used here.
52
Fig 12: Results of DNA Binding Assays

A) Representative EMSA gels assaying for DNA binding for WT and M8. Black triangle
indicates increasing protein concentration. Locations of DNA alone and DNA-protein
complexes are indicated. B) The curves of DNA binding for WT and M8. Error bars
represent the standard error of the mean. C) Summary of DNA binding data for all
mutants. Error bars represent standard error from curve fitting.

53
Fig 13: The results of helicase assays

A) Representative gel analysis result of helicase assay. B: boiled. UB: unboiled. DNA
positions for Y-DNA and ssDNA are indicated. B) Summary of the quantified helicase
activity of all mutants. Error bars represent standard error of the mean.

Discussion
The EXT-hp was previously shown to be important for helicase function based on
the study of a triple mutant on its tip (Brewster 2008). In this report, we follow up with a
systematic mutational and functional study in order to understand how this externally
located -hairpin immediately next to the exit of the side channels is involved in helicase
function by investigating the specific activities in oligomerization, DNA binding,
ATPase, and DNA unwinding.
The EXT-hp contains residues with hydrophobic, acidic, and basic properties.
There residues were mutated to alanine to examine the contributions of each of these
residues to the various activities associated with helicase function. A summary of the
data is listed in Table 5. Mutants M2-M4 contain mutations of hydrophobic residues. M2
54
Table 5: Summary of Biochemical Assays of the mutants
Mutant Oligomeric ATPase DNA Binding
No.* Mutant State ATPase Stimulation ssDNA Y-DNA Helicase
WT - Hexamer +++ +++ +++ +++ +++
M2 V324A Hexamer ++ +++ +++ +++ +++
M3 L325A Hexamer ++++ ++ ++++ +++ +
M4 VL324AA Hexamer +++ +++ ++++ +++ ++
M5 E326A Hexamer +++ +++ ++++ +++ +++
M6 D327A Hexamer ++ ++ +++ + ++
M7 ED327AA Hexamer ++ ++ + + +++
M1 R323A Hexamer ++ +++ ++ + +
M8 R329A Hexamer ++ ++ + + +
M9 R331A Hexamer 0 0 + + 0
M10 K346A Hexamer 0 0 +++ ++ 0

Mutations are grouped by residue type (green: hydrophobic, blue: acidic, red:
basic). +++: wild type activity level. ++++: greater than wild type (more activity or
tighter binding). ++: less than wild type. +: substantially less than wild type. 0: no
detectible activity. ATPase stimulation refers to the increase of ATPase activity upon
addition of Y-DNA. * Internally we have different numbers for these mutants as we re-
numbered them for publication. M1 is internally mutant 25, as it was added late. M2-
M10 are internally mutants 1 through 9. This chapter uses the numbering we submitted
for publication, not the internal numbers.

weakens ATPase activity while M3 increases it, simultaneously losing DNA stimulation.
The double mutant M4 cancels these effects. M3 has tighter DNA binding, with the
double mutant retaining this effect. It appears that the increasing ssDNA binding by
these mutants is associated with the reduction of helicase activity. At this moment, we
don’t have a good explanation for this observation. Probably tighter ssDNA binding by
M3 and M4 somehow inhibits efficient DNA translocation by these mutants.
Mutants M5-M7 contain mutations of acidic residues (Table 5). M5 showed a
slight increase in ssDNA binding, but no effect on other activities. M6 revealed weaker
ATPase activity, Y-DNA binding and helicase activity. It is interesting that the aspartate
55
mutation could decrease DNA binding. The puzzling data in this set, however, is the
double mutant, M7. Despite the weaker ATPase activity and DNA binding, it showed
near wild-type helicase activity.
For the mutants involved the basic residues (M1, M8-M9), they have the most
profound effect in all the activities assayed. Reduced ATPase activity, DNA binding and
helicase activity are observed across the board. Of note, M9, which had been assayed for
ATPase and helicase activity previously (Moreau 2007), now is revealed to have
compromised DNA binding as well.
Our single and double mutant EMSA assays from the current round of
experiments reveal decreases in DNA binding on the order of 1.5 to 2x fold increases in
K
d
. Mutations in the PS1-hairpin resulted in a similar change in DNA binding (2x fold
increase in K
d
) (McGeoch 2005). Therefore, the EXT-hp appears to be involved in
interactions with DNA, as shown for as other three -hairpin structural elements in the
MCM structure.
Some of the specific changes in DNA binding caused by mutations appear to be
counter intuitive. For example M6 and M7, which are both changes eliminating negative
charges, have a lowered affinity for DNA. Also, the increase in binding from
hydrophobic mutations is interesting. Similarly, how two basic amino acids on opposite
sides of the hairpin (K323 and R329) could impact DNA binding in similar ways is
unclear. While we are tempted to speculate that the proximity of R329 (decreased DNA
binding, decreased helicase activity) to the putative side-channel implicates the hairpin in
pulling ssDNA through the channel in a side channel extrusion model (see (Brewster
56
2008)), there are other possibilities as well. One possibility is, as proposed by Rothenberg
et al. in a DNA exclusion model (Rothenberg 2007), that one ssDNA may be on the
exterior of the hexamer to interact with the EXT-hp while the other ssDNA is
translocated in the central channel.
It is important to note here that decreases in DNA binding seem correlated with
decreases in ATPase activity. The DNA binding defects could be explained by
conformational changes from ATP binding that are not possible in the mutations, which
changes diminish the ability of the rest of the enzyme to bind DNA. Thus these DNA
binding defects could be secondary effects. However, it could easily be argued the other
way around. DNA binding stimulates ATPase activity, so mutations with DNA binding
phenotypes could also result in slowing the rate of ATP hydrolysis as a secondary effect.
This is at least partially borne out by the fact that the k
m
’s for ATPase activity do not
seem unduly affected by the mutations, with the exceptions of M9 and M10 (Table 4).
Thus, the enzyme may be still binding ATP with wild-type affinity, but hydrolyzes ATP
slower due to problems with DNA binding.
Finally, R331 is a residue that has fascinated us since we first examined the
ssoMCM structure and compared it to known mutations (Moreau 2007). As part of an
examination of sequence alignments and structure alignments of ssoMCM with viral
hexameric helicase LargeT antigen (LTag), we discovered that R331 align quite well
with LTag K418 that serves as a “lysine finger” and, in combination with LTag’s
arginine finger, coordinates the ATP gamma phosphate in the ATP binding and
hydrolysis (Li 2003, Gai 2004). K418 is vital for LTag ATPase and helicase activity
57
(Greenleaf 2008), which mimics the phenotype we see here. Thus, the base of the EXT-
hp seems directly involved in ATP hydrolysis.
The biochemical analysis we present here further establishes the EXT-hp as vital
for the activity of the MCM helicase. Such a role in helicase function probably is
associated mainly with the DNA binding activity of this EXT-hp. How this EXT-hp
interact with DNA and how such interactions are related to unwinding activity will
require further structural and functional analysis in the future.
58
Chapter 4: Modeling the DNA unwinding activity of MCM helicase
Adapted from a manuscript currently in revision.

In this chapter, several aspects of MCM modeling are presented that are inferred
from the structure and function based studies in the previous chapters.
Oligomerization of MCM
While it is well accepted that MCM forms hexameric complexes, it is unclear if
MCMs from different organisms will form double hexamers. Fortunately, some parallels
exist between what is already observed for SV40 LTag and MCM. Currently, there is a
large amount of evidence supporting a double hexamer as the active helicase for SV40
LTag for viral DNA replication (see (Sclafani 2004) and references therein). Further,
double hexamerization of LTag greatly stimulates helicase activity (Smelkova 1998,
Alexandrov 2002), and is generally important for viral DNA replication (Mastrangelo
1989, Smelkova 1997, Simmons 2000). As SV40 relies on cellular co-factors, it is
conceivable that this process used by LTag would mimic that of MCM’s in eukaryotic
cells. In eukaryotes, the hexamer composed of two MCM4, 6 and 7 trimers has also been
shown to be more processive as a double hexamer than a hexamer, although it is unclear
whether the two hexamers were in a head to head configuration in that report (Lee 2001).
While much less is known about the active form of the MCM helicase during
genomic replication in archaea and eukaryotes, crystal structure and EM studies from our
group and others reveal a double hexamer architecture for mthMCM (Fletcher 2003,
Fletcher 2005, Gómez-Llorente 2005, Costa 2006). Further, recent work has established
59
that the MCMs from S. cerevisiae (scMCM) are loaded as a double hexamer onto origin
dsDNA (Remus 2009). The emerging data supporting a double hexameric structure of
replicative helicases in archaea and eukaryotes may suggest a unified theme for a
functional double hexamer in vivo, despite the fact that details describing how a double
hexamer works to unwind two replication forks bi-directionally still remain largely
unknown (Sclafani 2004).
Double hexamerization of mthMCM is well studied, by X-Ray Crystallography
(Fletcher 2003) and by cryo-EM, with sample conditions in the presence and absence of
DNA. MthMCM’s N-terminal domains come together in a “head to head” configuration
(Fletcher 2005, Gómez-Llorente 2005, Costa 2006). This double hexamerization appears
to affect helicase activity in vitro, because the mutation of an N-terminal arginine
(R161A) at the double hexamer interface prevents formation of double hexamers and
weakens helicase activity, especially at lower protein concentrations (Fletcher 2005).
This is reminiscent of the case in SV40 large T antigen, where the double hexamer has
~10-15 fold higher helicase activity than the single hexamer (Smelkova 1997). However,
a recent report shows that mthMCM’s oligomerization state was concentration dependant
in the absence of nucleotide and DNA (Shin 2009). Lower protein concentrations yield
more monomers, higher concentrations favor hexamers, and even higher concentration
favor double hexamers. SsoMCM has so far been mainly reported as hexamers
(Carpentieri 2002), with a hint of evidence indicating larger oligomers, some of which
may contain double hexamers (Barry 2007) We also have some data indicating ssoMCM
60
double hexamerization may be salt dependent (Brewster et. al., in preparation). Thus,
double hexamerization of archaeal MCM may be dependent on local conditions.
Some parallels probably exist between what is observed for SV40 LTag and
MCM. Electron microscopy data of LTag-dsDNA unwinding complex shows a
population of “rabbit ear” structures indicative of bidirectional fork unwinding localized
to a single dodecameric complex (Wessel 1992). The population of these double loop
structures vs. bubble structures can be varied. For example, a monoclonal antibody was
added that theoretically bound to both hexamers. It was argued this stabilized the double
hexamer, and correspondingly it raised the population of rabbit ear loops. Conversely,
incubating with ETDA decreased the population. Local conditions can thus affect double
hexamer formation and cofactors could stabilize it, creating the rabbit ears structure.
Whether MCM operates as a single or double hexamer in vivo directly affects
how DNA replication is organized in the cell (reviewed in (Cook 1999, Sclafani 2004,
Takahashi 2005, Sakakibara 2009)). A single hexamer model implies two replication
forks traveling away from each other, centered around the origin of replication. In this
model, the DNA would be stationary and large replication machinery, including a large
body of co-factors, accessory clamps, primase and polymerase, would travel along the
DNA. Alternatively, if the MCM helicase operates instead as a double hexamer, the two
growing replication forks would be held close to each other by the double hexamer
helicase, which would act as an anchoring platform for assembling other replication
proteins on the growing forks. Rather than a large replication protein complex moving
along the DNA, the chromosomal DNA would be pulled into the replication complex that
61
may be anchored on the nucleo-matrix. Currently, evidence is building that the latter
model of stationary factories is the favored in vivo model (Cook 1999, Anachkova 2005).
62
Fig 14: Possible DNA unwinding modes by MCM helicase.

(A), (B) Schematic representation of MCM helicase as a hexamer (A) or double hexamer
(B). The 4 β-hairpins (NT, H2I, PS1, and EXT hairpins) are represented by short solid
bars; the central channel and the side channels are in darker shades. (C), (D) Steric
exclusion model for a single hexameric (C) or double hexameric (D) MCM helicase. (E)
Double side extrusion model, showing ssDNA exiting from two different side channels.
(F) Double side extrusion model adapted to a double hexamer becomes the looping
model. (G) Single side-channel extrusion model, showing one ssDNA strand extruding
from a single side channel, while the other ssDNA strand remaining in the central
channel. (H) Asymmetric looping model, ssDNA is extruded from N- and C- terminal
side channels. DNA is shown as black lines. Arrows indicate direction of DNA
translocation movement. Figure and caption adapted from (Brewster 2008).

63
Possible models of DNA unwinding by the single hexamer
While it is still in debate which hexameric state is the biologically active form for
cellular DNA replication in vivo, it is clear that a single hexamer of MCM, as well as
LTag, can unwind DNA substrates with 3’-end ssDNA overhangs in in vitro assays. This
is similar to the case for the DnaB family helicases in prokaryotic cells and phages, which
require a 5’-end ssDNA overhang to unwind the dsDNA substrate. In this regard,
substantial biochemical evidence has been gathered indicating probable sites of DNA
interaction in ssoMCM and mthMCM, which can help elaborate models for MCM-DNA
interactions during unwinding using dsDNA substrates with ssDNA available as the 3’-
overhang. Currently, there are several unwinding models that are proposed for MCM as a
single hexamer: the steric exclusion model, the rotary pump model, the strand extrusion
model, and the plowshare model.
Steric exclusion
Steric exclusion is proposed as the unwinding mechanism for the DnaB family
helicases in prokaryotes, which likely work as a single hexamer for genomic DNA
replication (Patel 2000). In this model, a helicase hexamer encircles and translocates
along ssDNA towards the fork, unwinding the DNA fork by excluding the opposite
strand from the hexamer (Fig 14C). The MCM 4, 6, 7 hexamer has been shown to use
this mechanism on synthetic fork substrates in vitro (Kaplan 2003). Further, the viral E1
helicase ssDNA bound crystal structure indicates this may be one of the unwinding
mechanisms for E1 (Enemark 2006). Finally, FRET data seems to show that the 5’ tail in
a forked substrate dynamically interacts with the exterior of the ssoMCM protein, rapidly
64
binding and unbinding during unwinding (Rothenberg 2007). This mechanism is
conceptually operable for any single hexameric helicase.
Rotary pump
Another model is the rotary pump, wherein many single hexamers bind at an
origin, spread out along the chromosome, then are anchored in place and rotate DNA—
thus, introducing negative supercoiling to unwind it (Takahashi 2005, Sakakibara 2009).
This model is proposed partially based on the observation that large numbers of excess
MCM are loaded during replication on the chromosome (reviewed in (Forsburg 2004)).
Considering the large number of replication proteins and chromosomal associated
proteins bound to the DNA, it would require a large scale coordination between these
proteins for this mechanism to work.
Strand extrusion and plowshare
Two reports analyzed DNA binding to ssoMCM mutants consisting of only the N-
terminal or C-terminal domains. The first report found that N-ssoMCM binds ssDNA
with an affinity at least comparable to wild type but that C-terminal ssoMCM (C-
ssoMCM) has greatly compromised ssDNA binding (Pucci 2007). The second report
found that C-ssoMCM can bind dsDNA with good affinity (and in a highly cooperative
manner), but N-ssoMCM cannot (Liu 2008). The N-ssoMCM structure bears this out, as
the central channel seems too narrow to encircle dsDNA (Liu 2008). However, the N-
terminal double hexamer of mthMCM clearly can bind dsDNA and ssDNA (Fletcher
2003). Interestingly, an EM study of mthMCM in the presence of dsDNA reveals dsDNA
in the channel bound to one of the hexamers in the double hexamer, and possibly only in
65
the C-terminal (Costa 2006). Together these data all seem to indicate that the hexamer
can accommodate both dsDNA and ssDNA in the C-domain and the N-domain, but may
possess a preference for dsDNA in the C-domain and for ssDNA in the N-domain.
During DNA unwinding by a single hexamer, if the C-domain binds dsDNA and the N-
domain binds ssDNA, then the other ssDNA strand must be extruded between the two
domains through the side channel, which leads to the strand extrusion model (Fig 14G).
In this model, one DNA strand is extruded through one of the six side channels
near the ATP binding site, while the other strand continues in the central channel and
exits through the N-terminal channel. Some of the earliest work on archaeal MCMs
found that the ssDNA binding site and the ATP binding site may overlap (Chong 2000).
This could be due to conformational shifts upon ATP binding directly affecting ssDNA
affinity. However, it may also be due to the proximity of the ATP binding site to the side
channel that may serve to passage ssDNA.
Extruding both strands out of two side channels (Fig 14E) could be an alternative
unwinding mode. However, this model does not seem to agree with the findings that the
interaction of the N-terminal NT-hp with DNA is important for helicase activity (Fletcher
2003, McGeoch 2005). At this moment, it requires further investigation as to whether this
interaction of NT-hp with DNA is important only for the initial DNA binding at the
beginning of unwinding or critical during the actual unwinding as well.
A previously proposed model for DNA unwinding by a rigid motif, the
“plowshare” (Takahashi 2005), can be adapted to the strand exclusion model. The
plowshare was thought to be at the end of a single hexamer with respect to the direction
66
of dsDNA movement and would be dragged behind the hexamer as it translocated on
dsDNA, splitting the two strands during the translocation process in a manner similar to
the RecBCD pin (Singleton 2004, Takahashi 2005). Such a pin is not present at the exit
of the central channel in our ssoMCM hexamer structure model. While the NT-hp is
situated near a likely DNA exit pore (the N-domain central channel), it is dispensable for
helicase activity (McGeoch 2005, Barry 2009). Thus it was proposed that the H2I-hp in
the middle of the channel may fill the role of the plowshare (Jenkinson 2006). The
presence and location of the H2I-hp is confirmed in the crystal structure of the near full
length ssoMCM (Brewster 2008). Furthermore, the crystal structure reveals an
unexpected (acidic) EXT-hp that is situated on the exterior of the protein near the side-
channel, not at the central channel. It is intriguing that EXT-hp is critical for ssoMCM
helicase function (Brewster 2008), and involved in DNA binding {Brewster et. al., in
preparation) which suggests that the side-channel may be used for DNA unwinding,
probably for direct ssDNA binding, as shown in model Fig 14G. The exact mechanism
for the involvement of this EXT-hp in DNA unwinding is worth further investigation.
The model in Fig 14G is attractive in that it satisfies all of the DNA binding data
discussed above, including the encircling of dsDNA by MCM that likely is biologically
relevant (reviewed in (Takahashi 2005)), and the involvement of each of the 4 hairpins in
binding DNA (see discussion below). However, it runs afoul of an unwinding result
obtained in vitro using fork DNA or ssDNA overhang dsDNA substrate, in which MCM
4, 6, 7 heterohexamer cannot unwind substrate with a biotin/streptavidin block on the 3’
 5’ strand, but can unwind substrate with a block on the 5’  3’ strand (Kaplan 2003).
67
This result argues for the strand exclusion model in Fig 14C. One caveat of these results
is that the unwinding experiment used dsDNA substrate with free 3’ ssDNA overhang
and not from the true dsDNA origin on a blunt-ended dsDNA. An unwinding assay
system that can initiate melting and unwinding from origin dsDNA sequence from a
blunt-ended dsDNA substrate in vitro would likely assay the unwinding mode that is
more closely related to the in vivo situation during DNA replication. As an aside, we
note that the body of research assays helicase activity using small forked substrates. One
can easily argue that a forked DNA unwinding assay is merely a translocation assay once
the hexamer is allowed to latch onto a ssDNA overhang. A more biologically relevant
helicase assay substrate should be a blunt ended dsDNA with an origin sequence in the
middle, and the MCM beginning its unwinding from the origin sequence in the middle of
the dsDNA. While no such assay system is available for MCM yet, SV40 LTag can
robustly unwind blunt ended dsDNA with an origin sequence in the middle (Bullock
1997). Nonetheless, the recent progress in being able to assemble a double hexamer of
scMCM on dsDNA may very well render such an assay system feasible for MCM soon.
Models of DNA unwinding by the double hexamer
As discussed above, various structural and biological data suggest that SV40
LTag works as a double hexamer for viral DNA replication in vivo, and available
evidence also suggests that MCM may function as a double hexamer. If these ring-
shaped helicases function as a double hexamer to unwind two replication forks bi-
directionally, how do the two DNA forks propagate and how are they organized by the
68
double hexamer? We present several hypothetical models and discuss how they fit the
present body of research data.
Steric exclusion
Simply expanding the model for a single hexamer in Fig 14C to a double hexamer
is impossible, as it would lead to non-complementary ssDNA strands sliding across one
another in the N-terminal central channel. However, if a side channel is utilized for the
ssDNA exit so that the N-domain no longer binds DNA anymore (Fig 14D), then the
steric exclusion model can be adapted to a double hexamer. Interestingly, this satisfies
many of the observations made above with regards to the single hexameric strand
extrusion model, but it does not engage the NT-hairpin. However, in this model, the NT-
hp could be utilized during initial melting and/or loading of the complex. This is seen in
S. cerevisiae MCM5; a triple mutant on the NT-hp affects replication initiation and origin
binding (Leon 2008, Bochman 2009). It also suggests a different role for the H2I-hp than
a plowshare: as the deletion of the H2I-hp increases DNA binding (Jenkinson 2006),
perhaps it serves to direct DNA in the central channel towards a side channel, where it is
picked up by the PS1-hp and extruded.
Looping models
A double looping model (Fig 14F) has been proposed for LTag previously (Li
2003, Gai 2004). This looping model can be directly extended from the unwinding
model for a single hexamer shown in Fig 14E. Again, the NT-hp is not utilized for DNA
unwinding, but perhaps initial N-terminal DNA binding is necessary for establishing
unwinding conformation, with DNA relocating to the side-channel after initiation and
69
conformational rearrangement. Because the AAA+ helicase domain alone of ssoMCM
showed helicase activity, this suggests that the N-domain is dispensable for unwinding,
which is consistent with this model. However, evidence also seems to suggest that
dsDNA and/or ssDNA interactions within the N-terminal central channel are important
for mthMCM beyond initiation (Fletcher 2003, McGeoch 2005). More investigation will
be needed to understand the N-terminal role in DNA unwinding in vitro and in vivo.
Regardless, the cases of dsDNA or ssDNA present in the N-terminal central channel can
be examined.
If dsDNA moves through the N-terminal domain of one of the two hexamers
during unwinding, then an odd situation of a double hexamer generating a single
replication fork arises (model not shown). It is noteworthy that mthMCM EM models
bound to dsDNA show an asymmetry lengthwise through the double hexamer (Costa
2006). One possibility is that one hexamer translocates on dsDNA behind the DNA fork
while the other hexamer at the fork separates it and extrudes ssDNA, leading to this
asymmetry in the structure. In this case, instead of two single hexamers moving at two
forks, or one stationary double hexamer holding two separate forks together, there would
be two double hexamers, possibly mobile, each with a single fork. However, given the
double hexamer architecture observed in SV40 large T, MCM in archaea and eukaryotes
(Gómez-Llorente 2005, Valle 2006, Remus 2009), the model of one double hexamer on
one replication fork is less likely, although we cannot exclude it. We note here that
recent work has shown that eukaryotic MCM double hexamers can at least “slide” along
dsDNA (Remus 2009).
70
If ssDNA moves through the N-terminal domain, as proposed during unwinding
for a single hexamer shown in Fig 14G, it can be easily adapted to the unwinding by a
double hexamer (Fig 14H). ssDNA of the two opposing strands would pass through part
of the central channel and then loop out from N- and C-terminal domain side channels
observed in crystal structures of N-mthMCM, as well as in the hexamer model of
ssoMCM (Fletcher 2003, Brewster 2008), which is a model that is reminiscent of past
looping models proposed or SV40 large T (Gai 2004, Valle 2006). Here, one DNA
strand is extruded out of a C-terminal side channel, and the other is extruded out of a
putative N-terminal secondary side channel. Secondary N-terminal side channels can be
visualized in the EM structure of double hexameric LTag (Valle 2006). Further, these
channels are also visible in mthMCM EM maps (Pape 2003) (Fig 15), and the recent
eukaryotic EM MCM map (Remus 2009). Additionally, they are also visible in the
hexamer model generated from the X-ray structure of ssoMCM. In this model, parts of
both C-terminal central channels contain dsDNA, and both N-terminal central channels
contain ssDNA, and all the functionally important structural motifs in the ssoMCM
structure are engaged. As such, this unwinding model by a double hexamer seems to
satisfy the structural and DNA binding data the most.
Detailed structural features for DNA unwinding
The ATP bound structure of LTag reveals a tight ATP binding pocket, with key
residues bonding with the ATP from both the cis- and trans- side (Gai 2004) (Fig 16, 17).
In the ssoMCM hexamer model, key trans- residues (such as the arginine finger) are

71
Fig 15: The side channels at the C-domain and N-domains

A) Electron-microscopy map of double hexamer mthMCM (Gómez-Llorente 2005) in
which a double hexamer model of ssoMCM fits. B) The EM map shown in panel A, but
with a higher threshold. The visible side channels in the N- and C- domains are
indicated.

within a reasonable distance, but are more recessed from the pocket, similar to, but to a
greater extant than the apo LTag hexamer structure (Gai 2004, Brewster 2008). Recent
work established that a residue at the base of the EXT-hp, R331, is critical for ATPase
and helicase activity (Moreau 2007). Is it possible that this residue has an analog in the
LTag binding pocket?
72
Fig 16: Overview of LTag hexamer conformations and the -hairpin structure in different
nucleotide binding states

Three nucleotide binding states of LTag hexameric helicase, seen from above (N-terminal
face). Monomers are colored, with their central β-hairpins in red (equivalent to the PS1-
hp of ssoMCM). The N-terminal domain, D1, has been removed for clarity. Adapted
from (Gai 2004).
In the sequence alignments of AAA+ ATPases including ssoMCM and LTag, a
stretch of 11 amino acids in ssoMCM or 5 amino acids in LTag in the C-terminal domain
do not align in the majority of the AAA+ ATPases (Fig 17A) (Iyer 2004). In ssoMCM,
these 11 amino acids are the EXT-hp. In LTag, the 5 amino acids form a small loop (Fig
17B). It appears that ssoMCM R331 may align with LTag K418. LTag’s K418, has
been identified as a “lysine finger” that works with the arginine finger to coordinate the
ATP gamma phosphate (Li 2003, Gai 2004). Mutation of LTag K418 to alanine
abrogates both ATPase and helicase activity (Greenleaf 2008).
73
Fig 17: The nucleotide pocket configurations in LTag and ssoMCM.
A) The sequence alignment between ssoMCM and LTag around the EXT-hp of
ssoMCM, adapted from (Iyer 2004). Secondary structure (H: helix, E: strand) is listed
above. The circled region is an 11 AA region (ssoMCM) or a 5 AA region (SV40 LTag)
that is not conserved among AAA+ ATPases. This region corresponds to the Ext-hp
(ssoMCM) or a small loop in LTag that houses its lysine finger (K418). The * indicates
ssoMCM R331 (top sequence) and LTag K418 (lower sequence). B) Structural alignment
of a portion of ssoMCM (green), and LTag (magenta). The 5 strand β-sheet that forms
the core of AAA+ ATPase is in the upper right; helix 0 from the C-domain is in the upper
left. C) LTag ATP binding pocket, with an ATP bound. Cis- residues (from the monomer
with the p-loop) are on the right from the blue monomer, trans- residues on the left from
the green monomer. The nucleotide pocket distance is indicated, measured from K418 to
K432 (main-chain to main-chain). D) SsoMCM nucleotide pocket from the hexamer
model, colored as in C. The pocket distance is measured from R331 to K346. Amino
acid side chains and the ATP position are modeled in.

74
Fig 17, continued

A careful structural alignment of apo LTag and the ssoMCM monomer reveals the
actual situation to be more complex (Fig 17B). The 5 strand beta sheets, ssoMCM’s Ext-
75
hp, and LTag’s 5 AA loop are shown. In terms of primary sequence, it appears ssoMCM
R331 is better matched with LTag K419, while ssoMCM R329 matches LTag K418 that
is called the Lysine finger. LTag K419 interacts with the ribose in the ATP bound
structure (Gai 2004), and has a much less severe phenotype than K418 (Greenleaf 2008).
Our triple mutant that included ssoMCM R329 also had a less severe phenotype than was
reported for ssoMCM R331 (Moreau 2007, Brewster 2008). While the k
cat
remained
unchanged, the k
m
for binding was significantly affected, and in the presence of Y-DNA,
the k
m
was high enough to make the k
cat
immeasurable. We have preliminary data for the
R329 mutant alone that shows a reduction, but not elimination of ATPase activity. Thus,
while the amino acids appear to be reversed in primary sequence, their phenotypes
suggest that ssoMCM R331 is equivalent to LTag lysine finger K418, and ssoMCM R329
is equivalent to LTag K419. This conclusion requires further verification with a structure
of ssoMCM hexamer bound to nucleotide.
In the ssoMCM hexamer model, we measured the main chain-main chain distance
of R331 (“lysine” finger) to K346 (Walker A) in angstroms. This “pocket distance” was
compared to the pocket distance of LTag (between equivalent LTag residues K418 and
K432), in both ATP bound and apo forms (Fig 17C, D). The LTag ATP bound pocket
distance is 12.6 angstroms compared to its apo distance of 18.4 angstroms. The ssoMCM
hexamer model has a pocket distance of 26.6 angstroms, which seems excessively large
for a residue that needs to interact with the ATP. If these two residues are directly
interacting with the ATP, it seems they need to close a 14 angstrom gap first.
76
Based on the discussion above and comparison with what is known about SV40
LTag, we propose a mechanism for conformational changes of ssoMCM in response to
the ATP binding cycle, which probably can couple the movement of the -hairpins for
DNA unwinding and translocation. First, ATP binding on the p-loop will bring R331 on
the EXT-hp of a neighboring monomer close to the ATP/p-loop to make bonding
contacts, closing the gap between the two monomers, and leading to the shift and rotation
of the C-terminal AAA+ domain relative to the N-terminal domain, very much like the
iris-movement observed in the SV40 LTag in response to ATP binding cycle (Fig 16, 18)
(Gai 2004). The N- and C-terminal domains have been shown to operate independently;
separately purified N- and C- terminal ssoMCM mixed together have stimulated helicase
activity compared to the C-terminal alone (Barry 2007). This domain shift and rotation of
the AAA+ domain should also lead to the coordinated movement of the three -hairpins
in the main and side channels, which can be coupled with DNA translocation and
remodeling (Fig 18). A movie simulating such conformational changes is shown as part
of the Fig 18 supplement.
Note, for the time being this movie is available at
http://aaron.brewsters.net/ratchet/ratchet.html

77
Fig 18: Nucleotide pocket closure in ssoMCM and the conformational changes of the
hexamer

A) Top and Side views of the ssoMCM hexamer model in the absence of nucleotide
binding (Brewster 2008). The N-terminal domain, as well as some surface loops, are
hidden for clarity. The three C-terminal -hairpins are colored. Red: helix-2 insert
hairpin (H2I-hp). Green: pre-sensor 1 hairpin (PS1-hp). Blue: exterior hairpin (EXT-
hp). The main-chain to main-chain distance from the EXT-hp residue R331 (“lysine”
finger) to the Walker A residue K346 is shown in angstroms (the “pocket distance”). B)
Same orientation shown as in panel A, but with the nucleotide pocket closure triggered
by ATP binding, resulting in the rotations and a translation of the C-terminal AAA+
domain of each monomer. See text for details.

Potential roles of the β-hairpins in DNA unwinding

In the simulation of the conformational changes triggered by ATP binding shown
in Fig 18, we modeled the tightening of the ATP binding pocket through a series of 5
steps, each involving 2 rotations and a radial translation. In each step, the axial rotation
78
was 5 degrees, the lateral rotation was 1.25 degrees, and the inward radial translation was
1 angstrom. The final pocket distance after these five step movements is 18.3 angstroms.
The end result of these movements is a tightening of the central channel, and an upward
movement (toward the N-terminus) of the PS1-hp and H2I-hp. These results parallel the
tightening of the LTag hexamer upon ATP binding (Fig 16) (Gai 2004).
The PS1-hp, required for helicase activity and involved in DNA binding in MCM and
LTag, lies recessed from the central channel in our hexamer model of ssoMCM. In our
simulation, closing the ATP pocket upon ATP binding seems to move the PS1-hp further
into the channel and up toward the N-terminus. If movement of this -hairpin into the
central channel upon ATP binding facilitates DNA binding, then perhaps hydrolysis of
ATP would restore the PS1-hp closer to the side channel, which could move separated
ssDNA with it and promote ssDNA extrusion through the side channel. Further, this
movement could cause a shift in NT-hairpin, mediated through the ACL, facilitating the
C- to N-terminal domain communication seen previously (Sakakibara 2008, Barry 2009).
Indeed, it was reported that the distance between the ACL and the PS1-hp (in trans-)
increases when ATP is bound (Barry 2009), which is supported by our modeling result.
The hexamer structure model of our crystal structure of ssoMCM shows that the
H2I-hp extends away from core subunit structure and protrudes into the central channel
(Brewster 2008). Further, the H2I-hp lies between two side channels and under the NT-
hp of an adjacent subunit, forming a helical arrangement of hairpins within the central
channel (Fig 4B-D). The simulation suggests that ATP pocket closure through ATP
binding may recess the H2I-hp away from central channel as the PS1-hp projects further
79
into it. ATP hydrolysis and release would restore the H2I-hp to the central channel,
which could split new base pairs along dsDNA. In this sense, it may act as a structural
element to insert into the dsDNA between strands in a plowshare manner as discussed
above. Indeed, while the H2I-hp itself is conserved, poor sequence conservation within
the H2I-hp has led to the idea that its interactions are non-amino acid specific,
presumably steric (Jenkinson 2006).
FRET data shows the 5’ tail of a forked DNA substrate binding and releasing to
the hexamer dynamically during unwinding, presumably to the exterior (Rothenberg
2007). The EXT-hp could be in a place suited to bind and disengage the 5’ strand from
the exterior of the protein. This would be accomplished through its hydrophobic and
acidic tip (VLED, 324-327). R329 and R331 would then be involved in ATP hydrolysis
and not DNA binding. However, our newly obtained analysis of this hairpin shows
E326, D327, R329 and R331 are all involved in DNA binding, making this theory
unlikely {Brewster et. al., in preparation). Further, in the ATP pocket closure model, we
see the EXT-hp moving directly around the exit of the side channel, a location suitable
for the engagement with DNA from the side channel.
These features all seem to suggest that the side-channel may be utilized for
ssDNA passage. If the strand separation occurs at the H2I-hp in the central channel near
the internal exit of the side channel, the separated ssDNA would be able to make its way
out of the hexamer through the nearby side channel. Thus, the unwinding models for a
single hexamer proposed in Fig 14E and 14G, or for a double hexamer in Fig 14F and
80
14H could all be likely modes of action for unwinding in in vitro experimental conditions
and in vivo replication.
The side channel paradox
In most of the unwinding models described in Fig 14, the side channels between
pairs of subunits are proposed as a possible route of extruding single stranded DNA
during unwinding. These side channels may simply be a byproduct of the structure
during hexamer assembly, and could play no functional roles. However, as discussed
above, the positioning of the important hairpins in the side channels, together with
mutational evidence showing the relevance of side channel residues for unwinding in
MCM (McGeoch 2005, Brewster 2008) and LTag (Wang 2007), gives tantalizing
evidence that the side channels may have be utilized for DNA unwinding. The paradox
is, there are six side channels per hexamer (12 in a double hexamer), but only one or two
of the six side channels will be used for ssDNA exit in these unwinding models.
We find a recent study on MCM using mutant doping experiments relevant to this
discussion (Moreau 2007). In these tests, wild-type and ATP hydrolysis mutants are
mixed in varying proportions and activity is measured. From these assays, it can be
determined if the 6 binding sites randomly hydrolyze ATP independent of each other
(probabilistic mechanism) or if there is a sequential mechanism, where one hydrolysis
event enables or stimulates the adjacent subunit’s ATP hydrolysis. In a probabilistic
model, activity falls off linearly as the %mutant increases, in a sequential model, one
mutated subunit will poison the entire hexamer and activity will fall off exponentially.
The data shows a semi-sequential model in this study for ssoMCM (Moreau 2007).
81
Specifically, up to three mutants can be tolerated, as long as the remaining three WT
subunits are sequential. This implies that only half of the hexamer is needed to unwind
DNA, which in turn means that only one or two side channels are necessary. Also, it may
imply that only 2-3 H2I hairpins are required to split DNA in a plowshare model.
If only a few ATP sites are necessary and only one side channel is needed, the rest
of the subunits may have no evolutionary pressure in terms of helicase function and can
evolve to specialize. This point may bear some relevance to what we see in eukaryotic
MCMs with six different subunits (MCM2-7): dimers of 3/7, 7/4 and 4/6 have ATPase
activity while the others do not (Davey 2003, Bochman 2008). Further, mutations in
Walker B or arginine finger in the 7/4 and 4/6 dimers affect helicase activity while the
same mutations in the other dimers have a smaller effect (see (Bochman 2009)). Finally,
the MCM 2/5 interface has been recently theorized to form a “gate” which both regulates
helicase activity and allows for loading onto dsDNA, thus not directly participating in
active unwinding (Bochman 2007, Bochman 2008). The 2/5 interface is likely opposite
the 7/4 interface (Davey 2003, Yu 2004), where ATPase and helicase activity seems to be
present.
Thus, one of these MCM dimers could be specialized to serve as a side channel
for ssDNA extrusion, such as the 7/4 side channel for example. Further, evolutionarily
specialized proteins could also allow consistent docking of accessory proteins, such as
kinase or polymerase, to a single subunit, allowing a specific arrangement of accessory
factors with respect to the side channel orientation, an advantage over the symmetric (or
non-differentiated) archaeal MCMs.
82
Conclusion
The wealth of new data regarding MCM structures and functions have
significantly aided the understanding of how MCM works in DNA unwinding and
replication. Here, we presented recent progress and insights into the structure and
functional relationship of MCM complex. Many of the detailed mechanisms remain to be
resolved by future studies, and clearly more data are needed to have a better
understanding of the unwinding by these replicative helicases in a cellular setting. We
hope the discussion here can serve as a basis for future studies of MCM functions in
DNA unwinding and replication.

83
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Appendix A: Cloning and purification of additional MCMs
Besides optimization of crystal growth, screening of cryo-conditions and other
crystal-handling techniques, often in x-ray crystallography the key to a high resolution
structure is to either change the construct, usually with a series of deletions to remove
disordered regions (see construct 612 from Chapter 2), or to change species entirely and
work with the equivalent enzymes from that species. In parallel with cloning and
subsequent crystallization efforts with construct 612, and in order to try and get higher
resolution structures, including that of the hexamer, I picked new MCMs to test in crystal
trials from 6 archaeal species with homology both near to and far from ssoMCM and
mthMCM. These archaea were Thermoplasma acidophilum (Tap), Sulfolobus
acidocaldarius (Sac), Metallosphaera sedula (Msd), Methanosarcina acetivorans (Mav),
Methanosarcina mazei (Mma), and Thermoplasma volcanium (Tvo). A full sequence
alignment is presented in Fig A1. From the alignment, the poorly conserved N-terminal
portion is revealed (lines 1 and 2), while the highly conserved MCM-box (lines 3-5) is
obvious, excepting some insertions in line 4.
With the help of undergraduate Matthew Tjajadi, we were able to clone these
constructs, either from frozen cells ordered from the ATCC catalog, or from genomic
DNA from the same catalog, into a pGEX vector and purify as described in Chapter 2,
with the exception of expressing them at 18-25ºC overnight. The resultant purifications
91
Appendix A continued
Fig A1: Sequence alignment of Archaeal MCMs

are shown in Fig A2. Note, we were unsuccessful at cloning sacMCM. Likely this is
because we had cells instead of purified DNA for our PCR template. Sulfolobus
acidocaldarius has an optimal growth condition of 80ºC+ and may simply not lyse at
higher temperatures. Several lysis procedures were attempted, but further work is
justified
Of the five successfully purified MCMs, only Tap yielded diffracting crystals
(crystallization done by post-doc Ganggang Wang), interestingly in a in a p61 space
92
Appendix A continued
Fig A2: Purifications of Archaeal MCMs
MW Marker Sso-FL Sso-612 Mav Mma Msd Tap Tvo

SDS page analysis of purification experiments. Sso-612: deletion construct used to
crystallize ssoMCM (see Chapter 2).

group which indicates the presence of filaments. Unfortunately, while the crystals
diffracted to 1.65 Å, it appears only the N-terminal domain is present in the crystal.
Nonetheless, phases were successfully determined using molecular replacement
techniques utilizing an ensemble of N-mthMCM and N-ssoMCM. Modeling is currently
underway.
93
Appendix B: 14 Additional MCM Mutants
Concurrent with the 10 mutations characterized in Chapter 3, I designed 14
additional mutations with the purpose of further characterizing MCM function. These
mutations are cloned and are being purified.
The mutations are summarized in Table B1. Figures B1 and B2 show alignments
of the cloned mutations (thanks to Susan Forsburg for these alignments). They can be
generally divided into three groups: ATP Pocket (mutations in residues that, based on
sequence and structure alignments with LTag, may effect ATP hydrolysis directly, or
couple that hydrolysis to DNA unwinding), functional (residues that seem “interesting”
from sequence alignments and structure comparisons), and side-channel (mutations
designed to either block, disrupt or alter the side channel, in an effort to investigate its
importance in helicase activity). Included in Table B1 are additional mutations that were
designed but not pursued and are included for completeness.
94
Appendix B continued
Table B1: Summary of SsoMCM Additional Mutations
Number* Mutation Location Category Notes Conservation
10 E422A Helix 3, exterior ATP Pocket Proposed to stabilize argine finger in LTag, is LTag D502 Absolute
11 H418A Helix 3 ATP Pocket Potential couplers of ATP hydrolysis to DNA unwinding Absolute
12 E419A Helix 3 ATP Pocket Potential couplers of ATP hydrolysis to DNA unwinding Absolute
13 HE418AA Helix 3 ATP Pocket Equivalent to LTag R498, D499 Absolute
14 R211A ACL loop Functional Directed at PS1-hp in trans Strong
15 K408A Central channel Functional Looking for arginines involved in DNA binding Absolute
16 R410A Helix 3 Functional Looking for arginines involved in DNA binding Weak
17 R414A Helix 3 Functional Looking for arginines involved in DNA binding Strong
18 DED411AAA Helix 3, interior Functional Mutate oddly acidic region Moderate
19 S206R ACL loop, at tip Side-channel Change charge of side channel Weak
20 G207R ACL loop, at tip Side-channel Change charge of side channel Absolute
21 T469R Helix 4 Side-channel Small 3-10 like helix (PPT) Weak
22 PP467AA Helix 4 Side-channel Small 3-10 like helix (PPT) Weak
23 V415A Helix 3 Side-channel Widen side channel Moderate
24 V415W Helix 3 Side-channel Narrow side channel Moderate
- R473A Arginine Finger Control Absolute
- DE404NQ Walker B Control Absolute
- QQ423AA Between H3, PS1-hp ATP Pocket Potential couplers of ATP hydrolysis to DNA unwinding Very strong
- R410E Helix 3 Functional Looking for arginines involved in DNA binding Weak
- R414E Helix 3 Functional Looking for arginines involved in DNA binding Strong
- DED411KRK Helix 3, interior Functional Mutate oddly acidic region Moderate
- S206A, F, W, D ACL loop, at tip Side-channel Further efforts to change the side channel Weak
- G207A, F, W, D ACL loop, at tip Side-channel Further efforts to change the side channel Absolute
- T469F, W, D, A Helix 4 Side-channel Further efforts to change the side channel Weak
- V415R, F, D Helix 3 Side-channel Further efforts to change the side channel Moderate
- Q348A, R, D, W, F Helix 1 Side-channel Near walker A, on the side channel Absolute

* see Chapter 3 for mutations 1-9 and 25. -: not cloned.
95
Appendix B continued
Fig B1: Sequence alignment: ACL Loop

** *
SsoMCM QERPEEVPSGQLPRQLEIILEDDLVDSARP 227
SpMcm2 QESPGTVPSGRLPRHREVILLADLVDVAKP 413
ScMcm2 QEAPGTVPPGRLPRHREVILLADLVDVSKP 420
XlMcm2 QESPGKVAAGRLPRSKDAILLADLVDSCKP 393
HsMcm2 QESPGKVAAGRLPRSKDAILLADLVDSCKP 408
DmMcm2 QESPGRIPAGRIPRSKDVILLADLCDQCKP 393
AtMcm2 QESPGTVPAGRLPRHKEVILLNDLIDCARP 426
ScMcm5 QELPDAVPHGEMPRHMQLYCDRYLCDKVVP 259
HsMcm5 QELPDAVPHGEMPRHMQLYCDRYLCDKVVP 259
XlMcm5 QESPDAVPHGELPRHMQLYCDRYLCDKVVP 260
DmMcm5 QELPDFVPQGEIPRHLQLFCDRSLCERVVP 254
AtMcm5 QENPEDVPTGELPRNMLLSVDRHLVQTIVP 256
SpMcm5 QEAPDMVPVGELPRHILLNADRYLTNQITP 255
SpMcm4 QETPDVVPDGQTPHSVSLCVYDELVDSARA 406
ScMcm4 QETPDFVPDGQTPHSISLCVYDELVDSCRA 429
XlMcm4 QESPGDMPAGQTPHTTILYAHNDLVDKVQP 379
HsMcm4 QESPEDMPAGQTPHTVILFAHNDLVDKVQP 384
DmMcm4 QESPDDMAAGQTPHNVLLYAHNDLVDKVQP 387
AtMcm4 QETPDEIPEGGTPHTVSLLLHDKLVDNGKP 352
SpMcm6 QENSNEIPTGSMPRTLDVILRGDIVERAKA 313
ScMcm6 QENANEIPTGSMPRTLDVILRGDSVERAKP 391
HsMcm6 QETQAELPRGSIPRSLEVILRAEAVESAQA 238
XlMcm6 QETQAELPRGAIPRSVEIILRAEAVESAMA 240
DmMcm6 QETQAELPRGCIPRAVEIILRSELVETVQA 232
AtMcm6 QETSKEIPAGSLPRSLDVILRHEIVEQARA 235
SpMcm3 QEMPERAPPGQLPRSIDILLDDDLVDTVKP 236
ScMcm3 QEMPEMAPAGQLPRSIDVILDDDLVDKTKP 288
XlMcm3 QEMPEKAPAGQLPRSVDIIADDDLVDKCKP 231
HsMcm3 QEMPEKAPAGQLPRSVDVILDDDLVDKAKP 231
DmMcm3 QEMPEKAPAGQLPRSVDIVCDDDLVDRCKP 228
AtMcm3 QEVPENAAPGQLPRSVDVIAEDDLVDSCKP 222
SpMcm7 QELTNQVPIGHIPRSLTVHLYGAITRSVNP 288
ScMcm7 QELSQQVPVGHIPRSLNIHVNGTLVRSLSP 345
AtMcm7 QELAEHVPKGHIPRSMTVHLRGELTRKVSP 261
XlMcm7 QEHSDQVPVWNIPRCMSVYVRGENTRLAQP 266
HsMcm7 QEHSDQVPVGNIPRSITVLVEGENTRIAQP 267
DmMcm7 QEHSDQVPVGHIPRSMTIMCRGEVTRMAQP 267

Species names are as given in Chapter 3 (Fig 10). *: mutated residue
96
Appendix B continued
Fig B2: Sequence alignment, ATP pocket and side channel mutations
* ****** ** * ***
SsoMCM IAVIDEIDKMRDEDRVAIHEAMEQQTVSIAKAGIVAKLNARAAVIAAGNPKFGRYISERPVSDNINLPPTILSRFDLIFIL 480
SpMcm2 VCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTTLQARCTIIAAANPIGGRYNTTIPFNQNVELTEPILSRFDILQVV 674
ScMcm2 VCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTTLQARCSIIAAANPNGGRYNSTLPLAQNVSLTEPILSRFDILCVV 683
XlMcm2 VCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTSLQARCTVIAASNPIGGRYDPSLTFSENVDLTEPIVSRFDILCVV 648
HsMcm2 VCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTSLQARCTVIAAANPIGGRYDPSLTFSENVDLTEPIISRFDILCVV 663
DmMcm2 VCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTSLQARCTVIAAANPIGGRYDPSMTFSENVNLSEPILSRFDVLCVV 648
AtMcm2 ICLIDEFDKMNDQDRVSIHEAMEQQSISISKAGIVTSLQARCSVIAAANPVGGRYDSSKSFAQNVELTDPILSRFDILCVV 681
ScMcm5 VVCIDEFDKMREDDRVAIHEAMEQQTISIAKAGITTTLNSRCSVLAAANSVFGRWDETK-GEDNIDFMPTILSRFDMIFIV 520
HsMcm5 VVCIDEFDKMREDDRVAIHEAMEQQTISIAKAGITTTLNSRCSVLAAANSVFGRWDETK-GEDNIDFMPTILSRFDMIFIV 520
XlMcm5 VVCIDEFDKMREDDRVAIHEAMEQQTISIAKAGITTTLNSRCSVLAAANSVYGRWDDTK-GEENIDFMPTILSRFDMIFIV 521
DmMcm5 VVCIDEFDKMREDDRVAIHEAMEQQTISIAKAGITTTLNSRCSVLAAANSIFGRWDDTK-GEENIDFMPTILSRFDMIFIV 517
AtMcm5 VVCIDEFDKMRPEDRVAIHEAMEQQTISIAKAGITTVLNSRTSVLAAANPPSGRYDDLKTAQDNIDLQTTILSRFDLIFIV 515
SpMcm5 IVCIDEFDKMRDEDRVAIHEAMEQQTISIAKAGITTILNSRTSVLAAANPIFGRYDDMKTPGENIDFQSTILSRFDMIFIV 512
SpMcm4 ICCIDEFDKMSDATRSILHEVMEQQTVTVAKAGIITTLNARTSILASANPIGSKYNPDLPVTKNIDLPPTLLSRFDLVYLI 685
ScMcm4 VCCIDEFDKMSDSTRSVLHEVMEQQTISIAKAGIITTLNARSSILASANPIGSRYNPNLPVTENIDLPPPLLSRFDLVYLV 708
XlMcm4 ICCIDEFDKMNESTRSVLHEVMEQQTLSIAKAGIICQLNARTSVLAAANPVESQWNPKKTTIENIQLPHTLLSRFDLIFLM 645
HsMcm4 ICCIDEFDKMNESTRSVLHEVMEQQTLSIAKAGIICQLNARTSVLAAANPIESQWNPKKTTIENIQLPHTLLSRFDLIFLL 650
DmMcm4 VCCIDEFDKMNDSTRSVLHEVMEQQTLSIAKAGIICQLNARTSILAAANPAESQWNKRKNIIDNVQLPHTLLSRFDLIFLV 652
AtMcm4 ICCIDEFDKMSDSARSMLHEVMEQQTVSIAKAGIIASLNARTSVLACANPSGSRYNPRLSVIENIHLPPTLLSRFDLIYLI 625
SpMcm6 ICAIDEFDKMDLSDQVAIHEAMEQQTISIAKAGIQATLNARTSILAAANPIGGRYNRKTTLRNNINMSAPIMSRFDLFFVV 616
ScMcm6 ICCIDEFDKMDISDQVAIHEAMEQQTISIAKAGIHATLNARTSILAAANPVGGRYNRKLSLRGNLNMTAPIMSRFDLFFVI 715
HsMcm6 VCCIDEFDKMDVRDQVAIHEAMEQQTISITKAGVKATLNARTSILAAANPISGHYDRSKSLKQNINLSAPIMSRFDLFFIL 536
XlMcm6 VCCIDEFDKMDLKDQVAIHEAMEQQTISITKAGVKATLNARTSILAAANPVGGRYERSKSLKHNVNLSAPIMSRFDLFFIL 538
DmMcm6 ICCIDEFDKMDQRDQVAIHEAMEQQTISIARAGVRATLNARTSILAAANPINGRYDRSKSLQQNIQLSAPIMSRFDLFFIL 528
AtMcm6 ICCIDEFDKMDIKDQVAIHEAMEQQTISITKAGIQATLNARTSILAAANPVGGRYDKSKPLKYNVNLPPAILSRFDLVYVM 535
SpMcm3 VVCIDEFDKMSDIDRVAIHEVMEQQTVTIAKAGIHTSLNARCSVIAAANPIYGQYDIRKDPHQNIALPDSMLSRFDLLFIV 496
ScMcm3 VVCIDEFDKMTDVDRVAIHEVMEQQTVTIAKAGIHTTLNARCSVIAAANPVFGQYDVNRDPHQNIALPDSLLSRFDLLFVV 549
XlMcm3 VVCIDEFDKMSDMDRTAIHEVMEQGRVTIAKAGIQARLNARCSVLAAANPVYGRYDQYRTPMENIGLQDSLLSRFDLLFIV 485
HsMcm3 VVCIDEFDKMSDMDRTAIHEVMEQGRVTIAKAGIHARLNARCSVLAAANPVYGRYDQYKTPMENIGLQDSLLSRFDLLFIM 485
DmMcm3 VVCIDEFDKMSDIDRTAIHEVMEQGRVTISKAGIHASLNARCSVLAAANPVYGRYDQYKTPMENIGLQDSLLSRFDLLFVM 480
AtMcm3 IVCIDEFDKMNDQDRVAIHEVMEQQTVTIAKAGIHASLNARCSVVAAANPIYGTYDRSLTPTKNIGLPDSLLSRFDLLFIV 475
SpMcm7 ICCIDEFDKMDESDRTAIHEVMEQQTISISKAGITTTLNARTSILAAANPLYGRYNPKVAPIHNINLPAALLSRFDILFLI 543
ScMcm7 ICCIDEFDKMDESDRTAIHEVMEQQTISISKAVINTNPGARTSILAAANPLYGRINPRLSPLDNINLPAALLSRFDILFLM 600
AtMcm7 ICAIDEFDKMDESDRTAIHEVMEQQTVSIAKAGITTSLNARTAVLAAANPAWGRYDLRRTPAENINLPPALLSRFDLLWLI 516
XlMcm7 VCCIDEFDKMMDTDRTAIHEVMEQQTISIAKAGIMTTLNARCSILAAANPAYGRYNPKKTVEQNIQLPAALLSRFDVLWLI 520
HsMcm7 VCCIDEFDKMAEADRTAIHEVMEQQTISIAKAGILTTLNARCSILAAANPAYGRYNPRRSLEQNIQLPAALLSRFDLLWLI 521
DmMcm7 VCCIDEFDKMADQDRTAIHEVMEQQTISIAKAGIMTTLNARVSILAAANPAFGRYNPRRTVEQNIQLPAALLSRFDLLWLI 521

Species names are as given in Chapter 3 (Fig 10). *: mutated residue
97
Appendix B continued
Fig B3: ATP Pockets of LTag and SsoMCM

A) ATP binding pocket for LTag (PDB ID 1SVM) (Gai 2004). B) SsoMCM ATP
binding pocket. Equivalent residues are colored the same in both views. One monomer
is shown in the background (light blue). Pieces of a second monomer providing residues
in trans- are shown in the foreground (light green) as well as the PS1-hp that may be
affected by ATP hydrolysis at this binding interface. In ssoMCM, side chains, where
shown, are modeled. The position of the ATP in ssoMCM is modeled based on LTag
ATP bound structure (1SVM).

ATP Pocket
Four mutations (10-13) were done to examine more carefully the ATP binding
pocket (Fig B3). E422 is equivalent to LTag D502, a residue theorized to stabilize the
positive charge from the arginine finger (Greenleaf 2008). H418 and E419 are equivalent
to LTag R498 and D499. These two residues, near the ATP binding pocket and the PS1-
hp in ssoMCM and LTag, when mutated in LTag lack helicase activity while retaining
ATPase activity (Greenleaf 2008). It was proposed that these residues couple ATP
hydrolysis to helicase activity, acting as a lever arm moving in concert with the hairpin
98
Appendix B continued
(see also (Gai 2004)). One of our non-perused mutations, QQ423AA, has been mutated
previously (Moreau 2007) and is relevant to this discussion. This mutation lost ATPase
and helicase activity (DNA binding was not assayed). Making the analogy to LTag, they
Fig B4: Side channel and functional mutations

Close up view of an ssoMCM hexamer, centered on the side channel. Two monomers
are in the foreground, the cis- monomer (Walker A, B and Sensor 1 residues are
considered in cis-) is on the right in green. The trans- monomer is on the left in cyan.
View from the exterior of the hexamer. The blue residues indicate functional mutations,
while the red residues indicate side-channel mutations.

99
Appendix B continued
also suggested these residues act as a trans- lever, sensing and coupling ATP hydrolysis
to unwinding. My proposal is that these facts are all related: ssoMCM HE418, 419 may
couple ATP hydrolysis to QQ423, 424, perhaps chaining it all to the PS1-hp, with
potential involvement from E422. It is worth noting that all of these residues are
absolutely conserved.
Functional Mutations
The second group of mutations (Fig B4) was designed based on alignments and
structure analysis. These residues either align strongly or have an intriguing aspect to
their structure. R211 (number 14), for example is on the ACL loop (see Chapter 2),
directed at the PS1-hp in trans-. It is conserved in all MCMs analyzed, except MCM4,
where it is constitutively replaced with histidine. Please note that while these mutations
were under construction, a paper came out specifically mutating residues on the ACL
(Barry 2009). The mutations were:
 E202A, E203A, and Q208A
 E199–R211  SerAspGly (named ∆ACL)
 I239–S249  SerAspGly
 Double mutant with the above two
 K246A, R247A, ∆ACL

Thus while R211 has not specifically been examined, it was affected in the ∆ACL
mutation, which should be taken into consideration when our assays are analyzed.
100
Appendix B continued
K408 (mutant 15) is simply an absolutely conserved lysine that seems directed
towards the central channel. Mutations 16-18, the so-called Mr. Ded sequence
(MRDEDR), should be considered as a group. This sequence is on the non-buried
portion of Helix 3, lining the side channel but more proximal to the central channel. It
houses a curious combination of positive and negative charges considering its proximity
to the central channel/side channel interface. R410 is only conserved in MCM5, while
R414 is only not conserved in MCM6. The conservation of the acidic DED411-413
seems directly related to the conservation of its flanking arginines; when all three acidic
residues are present, the two arginines are also (ssoMCM, MCM5). However, when the
three acidic residues dwindle two 2 or 1, the flanking arginines become less conserved.
Side Channel Mutations
These mutations (Fig B4) were chosen to try and disrupt possible side channel
function, presumably by blocking the channel with bulky or charged residues. When
assaying these mutants it will be especially important to validate hexamerization, as there
is great potential here to disrupt the oligomerization interfaces instead of merely blocking
the channel.
Two residues (mutations 19 and 20) were chosen on the ACL that appear to be
directed towards the side channel. These residues would also be affected by the ∆ACL
mutation mentioned above. S206 is not conserved among eukaryotes, but G207 is almost
absolutely conserved (Fig B1). Both have been mutated to arginine.
101
Appendix B continued
Helix 4 is a small 3
10
-like helix with the sequence PPT, on the trans- side of the
side-channel (mutations 21 and 22). Weakly conserved, it seems to offer a good target
for side channel disruption. Helix 3, as described above, parallels the side channel in
trans-. V415 is only moderately conserved, and serves as a good point to either widen
(V A) or narrow (V W) the side channel (mutations 23 and 24).
102
Appendix C: Homology modeling of eukaryotic MCMs
As described in Chapter 1, eukaryotic genomes contain 6 different MCMs genes,
whose products from a heterohexamer (MCM2-7). One of the benefits of having a
structure is the ability to do homology modeling, or “threading”, using the structure and
known sequence data to investigate differences between MCMs. It seems reasonable that
a large region that is only present in one eukaryotic MCM, and not in any other
eukaryotic MCMs, nor in archaeal MCM, would serve a non-helicase specific role. As
MCM is a target for de-regulation in carcinomas (see Chapter 1), identifying and
modeling regions with non-helicase function is of interest as they could also be regulation
targets or have other unknown functions. These regions can be deleted in a model
organism such as fission yeast and their phenotypes evaluated. This appendix describes
this modeling exercise and the design of several potential deletions. Once cloned into the
appropriate fission yeast plasmids, these deletions would be evaluated by our
collaborator, Susan Forsburg.
Homology modeling consists of inputting to a program such as Swiss Model
(Bordoli 2009) a PDB file containing the atomic coordinates of a reference protein, and
inputting a sequence alignment between the reference protein and a target protein. The
software uses the alignment to “thread” the target sequence into the reference structure.
Differences between the reference and the target are dealt with as best as possible:
deletions are attempted to be bridged and insertions are modeled. For more detail see
(Bordoli 2009).
103
Appendix C continued
Creating the best sequence alignment is the first step towards a good homology
model. First, modeling was attempted using the large sequence alignment provided by
Susan Forsburg (see Appendix B). This alignment contains all of the 6 MCMs from a
variety of species, including human, frog, fly, fission and budding yeast, and thale cress.
I used ClustalX to add the ssoMCM sequence to this alignment. I quickly realized this
was the wrong approach. While MCMs in the same group (such as MCM2) across
species are highly homologous, differences between the MCM groups (such as between
MCM2 and MCM3) are extreme. A giant alignment of all of them incorrectly
emphasized large differences and created what were likely invalid homology models.
Next, I tried simpler alignments between only two proteins, for example
SpMCM2 to ssoMCM. This approach, however, seems to emphasize inter-species
differences. The best alignments came from aligning ssoMCM to a group of MCMs,
such as ssoMCM to all of the MCM2’s from the above species. This yielded consistent
results that seemed to better approximate the “real” differences between archaeal MCMs
and eukaryotic MCMs, and between eukaryotic MCM groups. Then, the ssoMCM and
SpMCM2 alignment could be extracted and inputted it to Swiss Prot.
Interesting results were actually attained from all the MCMs except MCM2, and
are presented below. For each MCM, the proposed mutation is given first, then a figure
showing the homology model with the mutation colored green, the alignment of the
region being mutated, secondary structure prediction from PSIPRED, and any notes.
104
Appendix C continued
MCM3

Δ527-578: deletion between two helices of the α/β-α linker

Fig C1: MCM3 homology model

XlMcm3 494 DQEIADHVLRMHRYRTPGEQDGYALPLGCSVEIFA-------------TDDPNASDVTDQELQIYEKHDNLLHG---PRKNK--------SKIVSMQFIRKYIHVA
HsMcm3 494 DREISDHVLRMHRYRAPGEQDGDAMPLGSAVDILA-------------TDDPNFSQEDQQDTQIYEKHDNLLHG---TKKKK--------EKMVSAAFMKKYIHVA
DmMcm3 495 DQMISDHVVRMHRYRNPKEADGEPLSMGSSYADSL-------------S--FVSSSEEKKDTEVYEKYDALLHGK--SRQRH--------EKILSVEFMRKYIHIA
AtMcm3 484 DSMISEHVLRMHRYKNDR---GEAGPDGS----------------------LPYAREDNAESEMFVKYNQTLHGKKKRGQTH--------DKTLTIKFLKKYIHYA
SpMcm3 505 DRALSEHVLRMHRYLPPGVEPGTPVRDSLNSVLNVGATN--------AAGVSTENVEQEVETPVWETFSSLLHANARTKKKE----------LLNINFVRKYIQYA
ScMcm3 558 DRSISEHVLRTHRYLPPGYLEGEPVRERLNLSLAVGEDADINPEEHSNSGAGVENEGEDDEDHVFEKFNPLLQAGAKLAKNKGNYNGTEIPKLVTIPFLRKYVQYA
SsoMcm 488 DRELANYILDVHSGKSTKN-------------------------------------------------------------------------IIDIDTLRKYIAYA

PSIPRED PREDICTION RESULTS

SpMcm3 505 DRALSEHVLRMHRYLPPGVEPGTPVRDSLNSVLNVGATN--------AAGVSTENVEQEVETPVWETFSSLLHANARTKKKE----------LLNINFVRKYIQYA
Pred: HHHHHHHHHHHHH HHHHHHH HHHHHH HHHHHHHHHHHH HHHHHHHHHHH
Conf: 999999999886047766677742100112233201235 66555400333210037777778765303566665 56999999999999
Mutation: GGXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX *

Start at the TP as G is likely disordered (this is safer as glycine is more flexible). X
indicates deleted amino acids. GG: creates a 3 glycine linker to help folding.

* From the structure, LL is likely to pack against an alpha helix. Therefore, trim at the
KKKE.
105
Appendix C continued
MCM4

Δ186-196: delete a small loop between 1st and 2nd helices of domain A. E191 is
conserved in MCM4.

Fig C2: MCM4 homology model

SpMcm4 170 IASFRGFLRGFKKKYR--PEYRNELMPPPDAEQLVYIEALRNMRIMGLEI
ScMcm4 191 TTNFRNFLMSFKYKFRKILDEREEFINNTTDEELYYIKQLNEMRELGTSN
XlMcm4 158 KEKFQRFVQRFIDPLAK----EEENVG-LDLNEPIYMQRLEEINVVGEPF
HsMcm4 163 KENFQRFLQRFIDPLAK----EEENVG-IDITEPLYMQRLGEINVIGEPF
DmMcm4 165 KSKFKSFIMRFIDPSAE----QDEISENIDVNQPLYLQKLEEIHTLEEPY
AtMcm4 134 KSAIEMFVKHFREAREN----SDDLFR-----EGKYMVSIRKVIEIEGEW
SsoMCM 7 QIDYRDVFIEFLTTFKG------------NNNQNKYIERINELVAYRKKS

Mutation:
SpMcm4 170 IASFRGFLRGFKKKYR--PEYRNELMPPPDAEQLVYIEALRNMRIMGLEI
SpMcm4 170 GGGXXXXXXXXX

Conf: 9999999986332001-23322202556566626999999999718807
Pred: HHHHHHHHHHHHHHHH--HHHHHHHCCCCCCCCCHHHHHHHHHHHCCCCE
AA: IASFRGFLRGFKKKYR--PEYRNELMPPPDAEQLVYIEALRNMRIMGLEI

PEYRNEL is a low confidence predicted helix. Very low. Therefore still a possible
candidate for deletion.

106
Appendix C continued
MCM5

S301A: putative serine/threonine phosphorylation site on the N-C linker. Not in
ssoMCM, but semi-conserved in eukaryotes.

Fig C3: MCM5 homology model

ScMcm5 291 SSYIRVLGIQVDTDGSGR-SFAGAVSPQEEEEFRRLAALPNV
HsMcm5 291 SSYIRVLGIQVDTDGSGR-SFAGAVSPQEEEEFRRLAALPNV
XlMcm5 292 SSYIRVVGIQVDTEGTGR-SAAGAITPQEEEEFRRLAAKPDI
DmMcm5 287 APYMRVVGITVDSEGAGAISRYSNITSDEEEHFRRMAASGDI
SpMcm5 283 NPYIRVVGIQMDSNDGSK--STPLFSEEEEEEFLEISRTPNL
AtMcm5 286 QPYIRVVGLEDTNEASSR--GPANFTPDEEEEFKKFADSQDV
SsoMcm5 254 DIYMKVSSIEVSQKVLDE----VIISEEDEKKIKDLAKDPWI

Results from a yeast-specific phosphorylation site predictor. S: semi- conserved S/T in
eukaryotes.

>Sequence 33 amino acids
# netphosyeast-1.0a prediction results
# Sequence # x Context Score Kinase Answer
# -------------------------------------------------------------------
# Sequence 13 S IQMDSNDGS 0.863 main YES
# Sequence 17 S SNDGSKSTP 0.494 main .
# Sequence 19 S DGSKSTPLF 0.565 main YES
# Sequence 20 T GSKSTPLFS 0.821 main YES
# Sequence 24 S TPLFSEEEE 0.814 main YES
#
NPYIRVVGIQMDSNDGSKSTPLFSEEEEEEFLE # 50
%1 ............S.....ST...S.........
107

Appendix C continued
Mutation: both to alanine (double point mutation).
108
Appendix C continued
MCM5

Δ537-542: delete a poorly conserved insert on the linker between 2 helices of the α/β-α
linker. This mutation is low priority and was canceled, in favor of the above MCM5
mutation.

Fig C4: MCM5 homology model

ScMcm5 540 HVSALTQTQA--VEGEIDLAK
HsMcm5 540 HVSALTQTQA--VEGEIDLAK
XlMcm5 541 HLSARTQSSS--VEGEVDLNT
DmMcm5 537 HLSSNKSAPSEPAEGEISLST
SpMcm5 532 HTNLQESSET-LAIGEIPFDK
AtMcm5 535 HASANKFSDE--NTDSKEDNW
SsoMCM 499 HSGKS-------TKNIIDIDT

PSIPRED PREDICTION RESULTS
Conf: 512567642335566877999999
Pred: HH HHHHHHH
AA: HTNLQESSETLAIGEIPFDKFRRY
XXXXXX

Cancel this mutation for now.
109
Appendix C continued
MCM6

Δ348-398: delete a large N-C linker insertion, modeled to interact with the N-terminal A
domain (the accuracy of this is debatable). The insertion includes a modeled beta hairpin,
361-374, which is highly conserved. 380-387 is not conserved except in ScMCM
(modeled as loop before a conserved helix).

This alignment was altered by hand. See below. Also, several deletions are proposed
(deletions 1-4).

Fig C5: MCM6 homology model

Left and center, a homology model from the original ClustalX alignment. Left: deletion
1 in green; center: deletion 2 in green. Right: model using alternate alignment below,
deletion 4 in green. Note in the alternate alignment, the insertion is modeled as a
compact domain with a two strand β-sheet and a α-helix, instead of an extended
loop/helix/hairpin structure. This domain would nestle into the “waist” of the hexamer,
described in Chapter 2.

SpMcm6 328 PGVKPEAYRDSRNFGGRDA---DGVTGLKSLGVRDLTYKLSFLACMVQ---------PDDANDKS-------GADVRGDGSQGIEEQDE--FLQSLSQEE
ScMcm6 406 PGVKPSSTLDTRGISKTTEGLNSGVTGLRSLGVRDLTYKISFLACHVISIGSNIGASSPDANSNNRETELQMAANLQANNVYQDNERDQEVFLNSLSSDE
XlMcm6zy 254 PGVRAETSSRVGGREGYEA---EGVQGLRALGVRDLSYKLVFLACYVCP-----------------------TNPRFGGKDLHEEDMTAESIKNQMSVKE
HsMcm6 253 PGARAETNSRVSGVDGYET---EGIRGLRALGVRDLSYRLVFLACCVAP-----------------------TNPRFGGKELRDEEQTAESIKNQMTVKE
XlMcm6 255 GDARMETGAKVTGGEGFNS---EGVQGLKALGVRDLSYRLAFLACYVGA-----------------------TNPRFGGKDLREEDQTAESIKNQMTVQE
DmMcm6 247 VGTRAENSSRHKPGEGMD-----GVTGLKALGMRELNYRMAFLACSVQA-----------------------TTARFGGTDLPMSEVTAEDMKKQMTDAE
AtMcm6 250 PGERAECRRDSSQQKSSTAG-HEGVQGLKALGVRDLSYRLAFIANSVQI-----------------------ADGSRNTDMRNRQNDSNEDDQQQFTAEE
SsoMCM 242 SRAVFDIYMKVSS----------------------------------------------------------------------IEVSQKVLDEVIISEED
SsoMCM* 242 SRAVFDIYMKVSSIEVSQKVLDEVI----------------------------------------------------------------------ISEED

*Alternate alignment done by hand. IEEQDEFLQS in pombe is predicted to form a helix.
ClustalX aligned this section with ssoMCM IEVSQKVLDEVI, which is clearly a loop in
110
Appendix C continued
the structure (forming latter part of the N-C linker). Therefore, I shifted it over. Using
this alignment between SpMCM6 and ssoMCM resulted in the right-hand model above.

PSIPRED PREDICTION RESULTS

Conf: 5653111020124344444441110121423433313789998862356543344434443210455556664189989
Pred: HHEE EEEEE EEEEEEEEEE HHHHHHHHHH HHH
AA: PGVKPEAYRDSRNFGGRDADGVTGLKSLGVRDLTYKLSFLACMVQPDDANDKSGADVRGDGSQGIEEQDEFLQSLSQEE
Deletion 1: XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
Deletion 2: GGGXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
Deletion 3: GGGXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
Deletion 4: GGGXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

Deletions 1-3 are based on the original homology model. Deletion 2 seems better
than deletion 1. Deletion 1 is based on the secondary structure prediction, and tries to
avoid deleting in the middle of strands or helices. However, in the figure, the deletion
appears as if it is taken out of the beginning of the N-C linker, instead of the middle.
Deletion 2 attempts to use the homology model to delete the middle of the insertion.
Deletion 3 is a compromise, using some secondary structure prediction information to
avoid deleting the predicted helix, while still using the homology model.

However, remodeling based on the alternate alignment gives what is likely the
best answer. It uses the homology model to delete the modeled small domain by
adjusting deletion 2 to cover the modeled domain and delete a conserved aspartate, also
cutting the predicted helix a bit. Also, note that the insertion is 52 amino acids long.
More than 52 amino acids cannot be deleted or we risk the protein not folding right based
on the ssoMCM structure. Deletion 1 is 47 residues, deletions 2 and 4 are 52 residues.
We will go with deletion 4.
111
Appendix C continued
MCM7

Δ132-163: delete an insertion with a small hairpin or sheet. The insertion is positioned
after the last strand of domain A, before the loop with the double 3
10
-like helices which is
upstream of domain C. The insertion clashes w/ PS1-hp of mcm3 in the hexamer model,
abutting MCM3’s domain C, and reaching past its own ACL (allosteric control loop). It
would be interesting if this deletion improves MCM3/7 binding.

Conserved AAs: R132, P141, P147, R(K)152, F158, R(K/Q)159, S(T)162.

Fig C6: MCM7 homology model

Mcm3 is on the left. Mcm7 is on the right. It has been shown previously that MCM3/7
abut each other in the hetero hexamer (see (Forsburg 2004)).

XlMcm7 96 DALDVYIEHRLMMEQR-----------------GRDP---------NEMRDSQNQYPPELMRRFELYFKAPSS------------SKARVVRDVKADSIG
HsMcm7 97 DVLDVYIEHRLMMEQR-----------------SRDP---------GMVRSPQNQYPAELMRRFELYFQGPSS------------NKPRVIREVRADSVG
DmMcm7 97 DALDVYIEHRLMMESR-----------------TRNP---------MEQRDERNSFPSELMKRFEVGFKPLST------------EKAHSIREVKAQHIG
SpMcm7 123 EVLDVIMQQRVQRNE------------------NIDP--------------EHKGFPPELTRGYDLYFRPVTR-----------NKKPFSVRDLRGENLG
ScMcm7 137 DVLDVILNQRRLRNERMLSDRTNEIRSENLMDTTMDPPSSMNDALREVVEDETELFPPNLTRRYFLYFKPLSQNCARRYRKKAISSKPLSVRQIKGDFLG
AtMcm7 94 DDHDILMTQRADDG-------------------TDNP----------DVSDPHQQIPSEIKRYYEVYFKAPSK------------GRPSTIREVKASHIG
SsoMCM 94 EKVHVRIVG---------------------------------------------------------------------------IPRVIELRKIRSTDIG
* †

* This arginine in ssoMCM may be the conserved arginine at the †. In which case, no
need to delete it.

PSIPRED PREDICTION RESULTS

Conf: 888888877765312233444158811012467999826888752173229978869
Pred: HHHHHHHHHHHHH HHHH EEEEEEEEEE HHH
AA: EVLDVIMQQRVQRNENIDPEHKGFPPELTRGYDLYFRPVTRNKKPFSVRDLRGENLG
|<-- Insertion -->|
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
Deletion: GGGXXXXXXXXXXXXXXXXXXXXXXXXX
112

Appendix C continued
Note:
 Strand 1 of domain A, in both SpMcm7 and ssoMCM is correctly predicted to be
a strand by Psipred.
 Strand 2 of domain A, in ssoMCM is predicted to be a strand (as the structure
supports).
 Strand 2 of domain A, in SpMcm7, is predicted to be a long helix, with high
confidence (EVLDVIMQQRVQR).
Either the prediction is wrong, or mcm7 is different, and features a lone strand in domain
A, that parallels a helix. My proposed deletion does not chop into the helix, nor does it
delete the conserved arginine (which may be present in ssoMCM anyway, see the above
note).

Abstract (if available)

Abstract The mini-chromosome maintenance protein (MCM) complex is an essential helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. Here, I first discuss my 4.35Å crystal structure of the near full-length ssoMCM. The structure shows an elongated fold, with five sub-domains that are organized into two large N- and C-terminal domains. A near full-length ssoMCM hexamer generated based on the 6-fold symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) hexamer shows inter-subunit distances suitable for bonding contacts, including the interface around the ATP pocket. Four unusual beta-hairpins of each subunit are located inside the central channel or around the side channels in the hexamer. Additionally, the hexamer fits well into the double-hexamer EM map of mtMCM. Mutational analysis of residues at the inter-subunits interface and around the side channels demonstrates their critical roles for hexamerization and helicase function. I also present a series of 25 structure based mutations, 9 of 10 of which have been characterized by DNA binding, ATPase and helicase assays. Finally, modeling of ATP binding and hydrolysis based on the above evidence is presented.

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Asset Metadata

Creator Brewster, Aaron Samuel (author)

Core Title Structure and function of archaeal McM helicase

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Publication Date 04/27/2010

Defense Date 03/01/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag beta-hairpin,cancer,DNA replication,nucleic-acid motor,OAI-PMH Harvest,replicative helicase

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chen, Xiaojiang S. (committee chair), Bradforth, Stephen E. (committee member), Forsburg, Susan (committee member)

Creator Email aaron.brewster@usc.edu,aaronbrewster@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2958

Unique identifier UC1163293

Identifier etd-Brewster-3623 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-312812 (legacy record id),usctheses-m2958 (legacy record id)

Legacy Identifier etd-Brewster-3623.pdf

Dmrecord 312812

Document Type Dissertation

Rights Brewster, Aaron Samuel

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu