Biochemical mechanism of TopBP1 recruitment to sites of DNA damage

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Biochemical mechanism of TopBP1
recruitment to sites of DNA damage

by

Julyana Acevedo

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)
December 2015

ii

Copyright
Page

iii

Dedication

I dedicate this work to my family.

To my parents, Mauricio and Idalia Acevedo.

To Monica, Karla, Mauricio, and Alex.

iv

Acknowledgements

I
would
like
to
acknowledge
and
thank
the
many
people
who
helped
me
during
the

completion
of
my
work
at
USC.

I
would
like
to
thank
my
thesis
advisor
Dr.
Matt
Michael
for
the

many
helpful
comments
and
suggestions
on
my
project.
He
was
always
very
willing
to
take
the

time
not
only
to
carefully
look
at
my
data
and
provide
thoughtful
feedback,
but
also
to
provide

useful
advice
on
everything
from
presentations
to
how
to
work
with
frogs.

I
would
also
like
to

thank
all
members
of
my
committee,
Drs.
Oscar
Aparicio,
Susan
Forsburg,
Judd
Rice,
Lin
Chen,

and
Sean
Curran,
for
taking
the
time
to
offer
their
advice
and
support
throughout
all
these
years.

Dr.
Aparicio
and
Dr.
Forsburg
offered
me
their
kindness
and
encouragement
since
we
first
met,

especially
when
I
first
joined
the
program
and
needed
it
the
most.
They
allowed
me
to
do
a

rotation
in
their
labs
where
I
had
the
opportunity
to
meet
really
wonderful
people
and
learn
a

lot,
and
have
continued
over
the
years,
to
provide
helpful
suggestions
on
my
project
during
joint

lab
meetings.
Likewise,
I
greatly
appreciate
having
had
advices
from
Dr.
Rice
and
Dr.
Chen,
who

always
took
the
time
to
offer
their
insight
and
perspectives
on
my
work,
which
were
important

in
making
sense
of
some
confusing
aspects
of
it.

I
am
greatly
indebted
to
Dr.
Sean
Curran
for
not

only
becoming
part
of
my
dissertation
committee
on
very
short
notice,
but
also
for
providing

useful
comments
and
suggestions
on
how
to
better
present
my
work.

I
would
also
like
to
thank

all
past
and
present
Michael
lab
members,
especially:
Chris
Murphy,
Hovik
Gasparyan,
Melina

Butuci,
Frances
Tran,
Matthew
Wong,
and
Ashley
Williams
not
only
for
their
helpful
comments

and
suggestions,
but
also
for
being
great
people
to
work
with
and
good
friends.

I
would
also
like

to
thank
Anna
Skylar,
Alice
Landolph,
and
Ji-‐ping
Yuan
for
helping
me
with
many
things
and
also

the
many
other
friends
I
made
while
at
USC.

Lastly,
but
not
least,
I
would
like
to
thank
my
whole

family
who
have
always
supported
and
encouraged
me
in
everything
throughout
my
life.

My

parents,
especially,
have
been
my
example
in
hard
work,
dedication,
and
love
that
have

motivated
me
to
pursue
whatever
I
wanted
in
life
and
I
am
very
thankful
to
them.

v

List
of
Figures

Chapter 1

Figure
1.
Diagram
of
the
various
roles
of
TopP1
inside
the
cell
.......................................................
9

Chapter
3

Figure
2.
TopBP1
binds
ssDNA
in
an
RPA-‐
and
length-‐dependent
manner.
............................
33

Figure
3.
Deletion
analysis
of
TopBP1
binding
to
RPA-‐ssDNA.
.....................................................
35

Figure
4.
PTIP
BRCT2
binds
ssDNA
in
an
RPA-‐
and
length-‐dependent
manner
....................
37

Figure
5.
Functional
characterization
of
the
TopBP1
BRCT2
interaction

with
RPA-‐ssDNA
.................................................................................................................................................
38

Figure
6.
ATR
activity
limits
TopBP1
chromatin
recruitment
.......................................................
40

Figure
7.
Revised
model
for
ATR
activation
during
checkpoint
response
................................
41

Chapter
4

Figure
8.
TopBP1
4/5WR
mutant
leads
to
an
enhanced
checkpoint
response

that
cannot
be
explained
by
an
increase
in
DNA
replication.
.........................................................
57

Figure
9.
TopBP1
BRCT4/5
WR
mutant
does
not
enhance
checkpoint

complex
assembly.
.............................................................................................................................................
58

Figure
10.
TopBP1
BRCT4/5
WR
mutant
does
not
respond
to
ATR
activity

as
the
wild
type
and
shows
no
enhanced
recruitment
to
sites
of
damage.
..............................
60

Figure
11.
TopBP1
BRCT4/5
WR
mutant’s
ability
to
activate
the

checkpoint
is
not
dependent
on
damage
or
DNA
.................................................................................
61

Figure
12.
TopBP1
BRCT4/5
WR
mutant
ability
to
activate
the
checkpoint

is
enhanced
by
the
addition
of
a
phosphatase
inhibitor
...................................................................
62

Figure
13.
TopBP1
phosphorylation
status
in
response
to
damage
is

different
in
TopBP1
mutants..
......................................................................................................................
63

Figure
14.
TopBP1
BRCT4/5
WR
shows
possible
enhanced
oligomerization.
.......................
63

vi

Table
of
Contents

Title
Page
.....................................................................................................................................
i

Copyright
Page
..........................................................................................................................
ii

Dedication
................................................................................................................................
iii

Acknowledgements
................................................................................................................
iv

List
of
Figures
............................................................................................................................
v

Abstract
of
the
Dissertation
...............................................................................................
vii

Chapter
1:
Introduction
...................................................................................................................
1

1.1
DNA
Replication
...................................................................................................................................
2

1.2
Checkpoint
Response
.........................................................................................................................
3

1.3
ATR
signaling
.........................................................................................................................................
4

1.4
Essential
role
of
TopBP1
for
both
DNA
replication
and
checkpoint

establishment
in
response
to
DNA
damage
.........................................................................................
5

1.5

Overview
of
the
projects
presented
in
this
dissertation……………………………………..7

Chapter
2:
Materials
and
Methods
..................................................................................
10

Plasmids
...........................................................................................................................................................
11

Xenopus
egg
extracts
and
sperm
chromatin
isolation
.................................................................
12

Expression
vectors
and
IVT
protein
production
.............................................................................
12

Recombinant
proteins
................................................................................................................................
12

Replication
assays
........................................................................................................................................
13

ssDNA
binding
assays
.................................................................................................................................
13

GST
pull-‐down
assays
.................................................................................................................................
14

Immunodepletion
and
antibodies
.........................................................................................................
14

Chapter
3:
Direct
binding
to
RPA-‐coated
ssDNA
allows
recruitment

of
the
checkpoint
activator
TopBP1
to
sites
of
DNA
damage
.................................
16

3.1
Introduction
............................................................................................................................................
18

3.2
Results
........................................................................................................................................................
23

TopBP1
binds
ssDNA
in
a
length-‐
and
RPA-‐dependent
manner
..........................................................
23

TopBP1
uses
its
BRCT2
domain
to
bind
RPA-‐ssDNA
................................................................................
25

TopBP1
BRCT2
W265R
fails
to
accumulate
on
DNA
DSB-‐containing
chromatin

and
fails
to
activate
ATR
during
a
DSB
response
........................................................................................
27

ATR
regulates
TopBP1
association
with
chromatin
..................................................................................
29

3.3
Discussion
................................................................................................................................................
30

Chapter
4:
Analysis
of
TopBP1
structure
regulation
during
checkpoint

signaling
..................................................................................................................................
42

4.1
Introduction
............................................................................................................................................
44

4.2
Results
........................................................................................................................................................
47

A
TopBP1
4/5WR
mutant
leads
to
increased
checkpoint
signaling
...................................................
47

BRCT4/5
WR
does
not
hyper-‐accumulate
on
chromatin
in
the
absence
of
ATR

activity
and
can
activate
ATR
in
the
absence
of
DNA
damage
...............................................................
49

The
4/5
WR
mutant
is
able
activate
ATR
in
the
absence
of
DNA
.........................................................
50

4.3
Discussion
................................................................................................................................................
54

Bibliography
...........................................................................................................................
64

vii

Abstract
of
the
Dissertation

During
a
replication
stress
response
or
after
double
strand
break
resection,

the
ATR
protein
kinase
is
activated
to
allow
a
delay
in
cell
cycle
progression,
a
block

to
further
origin
firing,
and
stabilization
of
the
stalled
fork.

ATR
is
recruited
to

stalled
forks
via
an
interaction
between
its
binding
partner
ATRIP
and
RPA-‐coated

single-‐stranded
DNA
(ssDNA).

However,
binding
of
ATR-‐ATRIP
to
RPA-‐coated

ssDNA
is
not
sufficient
for
ATR
activation
as
both
the
9-‐1-‐1
clamp
protein
and
the

checkpoint
activator
TopBP1
must
also
be
recruited
so
that
TopBP1
may
activate

ATR.

The
biochemical
mechanism
for
assembly
of
the
checkpoint-‐signaling
complex

has
been
an
important
unanswered
question
in
the
cell
cycle
field
for
many
years.

Previous
work
from
our
laboratory,
and
other
groups,
has
shown
that

ATR/ATRIP
and
TopBP1/9-‐1-‐1
are
recruited
independently
to
stalled
forks,
and

then
come
together
on
the
DNA
to
form
an
active
checkpoint-‐signaling
complex.

Furthermore,
we
have
previously
shown
that
TopBP1
is
required
for
loading
of
the

9-‐1-‐1
clamp,
however
the
means
by
which
TopBP1
senses
the
stalled
fork
to
allow

checkpoint
complex
assembly
was
not
previously
known.

Our
data
shows
that

direct
interaction
between
TopBP1
BRCT
repeat
2
and
RPA-‐coated
ssDNA
is
the

mechanism
for
TopBP1
recruitment.
Importantly,
we
determined
that
direct

interaction
with
RPA-‐coated
ssDNA
is
a
general
feature
of
a
subclass
of
BRCT

domains
from
other
proteins,
such
as
PTIP.
Furthermore,
we
find
that
once
TopBP1

has
promoted
initial
ATR
activation,
further
chromatin
loading
of
TopBP1
to

damaged
sites
is
prevented
by
a
negative
feedback
loop
of
ATR
activity.

viii

Lastly,
we’ve
also
identified
and
studied
a
point
mutant,
BRCT4/5
WR,
that

seems
to
have
a
different
structure
to
the
wild-‐type
protein
and
strongly
enhances

the
checkpoint
in
the
absence
of
DNA
damage
or
even
DNA.

This
is
possibly
due
to
a

greater
accessibility
of
its
AAD
to
ATR,
but
further
work
is
needed
to
confirm
this
or

any
other
possibility.

Given
the
fact
that
this
mutant
strongly
enhances
the

checkpoint,
it
might
in
the
future
prove
useful
in
allowing
us
to
gain
a
clearer

understanding
of
the
changes
TopBP1
normally
undergoes
in
the
process
of

checkpoint
activation
and
might
be
important
in
further
describing
the
order
in

sequence
of
events
that
lead
to
checkpoint
activation.

1

Chapter
1

Introduction

2

1.0

Introduction

Accurate
transmission
of
genetic
information
relies
on
a
cell’s
ability
to
respond

to
and
properly
repair
damage
to
its
DNA.
What
a
cell
does
in
response
to
this

damage
can
have
major
consequences
not
only
to
the
survival
of
that
particular
cell

and
its
immediate
progeny
but
when
part
of
multicellular
organism,
to
the
organism

as
a
whole.
Accumulated
DNA
mutations
that
result
from
DNA
damage
can
affect

genomic
integrity,
which
can
lead
to
cancer.

DNA
damage
can
be
caused
by
any

number
of
environmental
factors
or
may
occur
spontaneously;
this
damage
may

result
from
replication
stress,
cellular
metabolism,
exposure
to
UV
light
or
ionizing

radiation,
or
harmful
chemicals.
In
order
for
a
cell
to
properly
respond
to
assaults
to

its
DNA,
it
must
signal
to
alert
and
then
tightly
coordinate
an
intricate
series
of

events
that
collectively
are
termed
a
checkpoint
response.
Depending
on
the

severity
of
the
damage
the
outcome
to
the
fate
of
the
cell
will
result
in
a
launch
of
a

checkpoint
response
that
will
organize
and
promote
repair
to
the
site
of
damage,
or

lead
to
apoptosis.

1.1
DNA
Replication

Duplication
of
the
genome
takes
place
during
the
synthesis
phase
(or
S
phase)

of
the
cell
cycle.

Before
reaching
this
point,
most
cells
undergo
growth
and
ensure

conditions
are
favorable
for
division
as
they
prepare
to
duplicate
their
DNA
(G1

phase).

At
this
point,
the
cell
establishes
origins
with
the
vast
majority
of
initiation

occurring
non-‐randomly
at
genetically/epigenetically
defined
origins
by
loading
of

3

origin
recognition
complex
(ORC)
and
making
them
into
pre-‐replication
complexes

(pre-‐RCs).
Pre-‐RC
formation
requires
the
recruitment
and
loading
of
cell
division

cycle
6
(cdc6),
chromatin
licensing
and
DNA
replication
factor
1
(cdt1),
and
the

mini-‐chromosome
maintenance
(mcm2-‐7)
complex
to
sites
bound
by
ORC.

During
S

phase
a
select
number
of
the
previously
established
pre-‐RCs
are
converted
into
pre-‐
initiation
complexes
(pre-‐IC’s)
and
origins
fire
to
begin
the
replication
process.
In

Xenopus
as
well
as
yeast,
in
order
for
pre-‐RC’s
to
be
turned
into
pre-‐IC’s,
both
cdc7-‐
drf1
and
cdk2-‐cyclin
E
activities
are
necessary
for
the
activation
of
the
replicative

helicase
(Pospiech,
2010;
Boos,
2011).
The
activity
of
cdk2-‐cyclin
E
leads
to
the

recruitment
of
cell
division
cycle
45
(cdc45).
Specifically,
cdk2-‐
mediated

phosphorylation
of
Treslin
(TopBP1-‐interacting,
replication-‐stimulating
protein)

allows
it
to
bind
to
TopBP1
(topoisomerase-‐2
binding
protein
1)
on
DNA,
this
then

allows
it
to
deposit
cdc45
onto
pre-‐ICs
(Van
Hatten,
2002;
Kumagai,
2010).
After
the

helicase,
or
cdc45-‐MCM10-‐GINS
(CMG
complex),
unwinds
DNA,
DNA
replication

occurs
(Im,
2009).

1.2
Checkpoint
Response

The
main
players
in
a
checkpoint
response
are
different
depending
on
the
type

of
DNA
damage.

Ataxia
Telangiectasia
Mutated
(ATM)
and
Ataxia
Telangiectasia
and

Rad3-‐Related
(ATR)
are
two
highly
conserved
kinases
between
organisms
that
are

part
of
the
phosphatidylinositol
3-‐kinase-‐related
kinases
(PIKKs)
protein
family.

PIKKs,
which
are

serine/threonine-‐protein
kinases,
are
related
to
phosphatidylin-‐
ositol
3-‐kinases
(PI3Ks)
that
are
also
key
components
of
signaling
pathways
in

4

response
to
different
stimuli.

While
ATM
is
almost
exclusively
activated
by
DNA

double
strand
breaks
(DSB),
ATR
is
mainly
activated
by
long
stretches
of
ssDNA,

most
commonly
present
during
S
phase
and
also
by
resected
double-‐strand
breaks

(Cimprich,
2008).

1.3
ATR
signaling

Polymerase
stalling
during
DNA
replication
can
occur
for
any
number
of

reasons,
such
as:
DNA
damage,
difficult
to
replicate
sequences
like
trinucleotide

repeats,
or
by
activated
oncogenes
(Cortez,
2001;
Batkova,
2005;
Zeman,
2014;

Hills,
2014).
Interference
of
replication
fork
progression
due
to
polymerase
stalling

can
lead
to
polymerase
and
helicase
uncoupling,
activating
a
replication
stress

response
(Burrows,
2005).
Replication
stress
activates
many
pathways
with
the

major
responder
being
ATR.
Activation
of
ATR
allows
it
to
activate
its
main
effector

protein
Chk1.
Chk1
activation
will
then
arrest
the
cell
cycle,
prevent
firing
of

replication
origins
near
damaged
sites,
and
recruit
proteins
to
the
damaged
site
for

repair.

Polymerase
and
helicase
uncoupling
results
in
generation
of
long
stretches
of

ssDNA
that
then
lead
to
RPA
coating
of
the
DNA
(Byun,
2005).
When
parts
of

chromatin
are
unwound
to
reveal
DNA
during
replication
or
damage,
RPA

immediately
coats
it
to
help
stabilize
it
and
prevent
breakage.
Each
RPA
hetero-‐
trimeric
protein
complex
is
composed
of
a
70kD,
32kD,
14kd
subunit
that
when

5

bound
to
DNA,
bind
and
protect
around
30
nucleotides
(Fan,
2012).
RPA-‐ssDNA
is

the
structure
recognized
to
allow
the
checkpoint
complex
to
form,
ultimately

activating
ATR
and
leading
to
Chk1
phosphorylation
(Ball,
2005).

ATR
is
brought
to
RPA-‐coated
ssDNA
by
its
binding
partner
ATRIP,
which

serves
as
a
bridge
between
the
two
(Ball,
2005;
Zou,
2003;
Choi,
2007;
Choi,
2010).

Yet,
ATR/ATRIP
localization
to
sites
of
damage
is
not
sufficient
to
promote
its
kinase

activity
(Kumagai,
2006a&b).

TopBP1
is
a
multi-‐BRCT
domain
protein
that
contains

an
essential
ATR
activation
domain
(Kumagai,
2006).
TopBP1
recruits
pol
alpha
and

911,
takes
them
to
the
site
of
damage,
where
pol
alpha
makes
a
primer
that
allows

Rad17
to
load
911
onto
the
5’
junction
and
mediates
assembly
of
the
checkpoint

(Yan,
2009).
TopBP1
associates
with
the
911
clamp,
which
leads
to
the
activation
of

ATR
(for
fuller
details
of
ATR
activation,
refer
to
section
3.1).

1.4
Essential
role
of
TopBP1
for
both
DNA
replication
and
checkpoint

establishment
in
response
to
DNA
damage

TopBP1
is
an
essential
protein
that
has
major
roles
in
various
processes
inside

a
cell
such
as
DNA
replication,
transcription,
and
the
establishment
of
the
DNA

damage
checkpoint
response
(Yamane,
1997;
Yamane
2002;
Makaneimi,
2001;

Tacarini,
2006;
Kumagai,
2006;
Sokka,
2010;
Germann,
2011;
Chowdhoury,
2011).

An
important
feature
of
TopBP1
is
that
it
contains
9
BRCT
repeats.
BRCT
domains

were
first
identified
in
BRCA1,
where
work
showed
they
are
able
to
mediate

protein-‐protein
interactions
(Yu,
2003;
Manke,
2003).

Further
studies
showed
that

6

these
domains
are
able
to
recognize
phospho-‐peptides
and
thus
distinguish
between

phosphorylated
and
un-‐phosphorylated
forms
of
their
binding
partners
(Yu,
2003;

Manke,
2003).

TopBP1
interacts
with
many
proteins,
for
example:
the
region
within

TopBP1
containing
BRCT0-‐2
is
necessary
for
binding
Rad9,
NBS1,
CtIP
and
Treslin

(Ramirez,
2012
;
Kumagai,
2010;
Lee,
2010);
the
linker
region
between
BRCT3
and
4

is
required
for
GINS
binding;
BRCT
4/5
bind
RecQ4,
MDC1(Wang,
2011;
Leung,

2013),
BLM
(Wang,
2013;
Wang,
2015;
Blackford,
2015),
53BP1
(Cescutti,
2010),

and
possibly
MRN
complex;
BRCT6
binds
E2F1
(Liu,
2003);
region
between
BRCT6

contains
an
ATR
activation
domain
(Kumagai,
2006);
BRCT7/8
bind
BACH1/FANCJ

(Leung,
2011),
p53
(Liu,
2009),
Miz
1(Herold,
2008),
and
facilitates
AKT
induced

TopBP1:TopBP1
oligomerization
(Liu,
2006).

As
mentioned
above,
TopBP1
interacts
with
many
proteins
and
this
results
in

its
association
with
chromatin
at
different
times.

There
are
three
modes
of
TopBP1

chromatin
association:
(A)
Mode
I:

association
with
replication
origins
during

replication
initiation
(B)
Mode
II:

cdk-‐dependent
coating
of
chromatin
during

unperturbed
S
phase
(function
unknown)
(C)
Mode
III:

association
with
sites
of

replication
stress
during
checkpoint
activation
(Yan,
2009)
(Figure
1).

During
DNA

replication
TopBP1
is
essential
at
the
initiation
step
to
allow
origin
firing.
TopBP1

recognizes
the
cdk2
phosphorylated
form
of
Treslin,
which
is
activated
once
cdk2-‐
cyclin
E
levels
rise
at
the
beginning
of
S
phase
(Kumagai,
2010).
This
ultimately

allows
the
loading
of
cdc45
by
Treslin/TopBP1
onto
the
pre-‐RC
and
allows
the

initiation
of
DNA
replication.

7

As
previously
mentioned,
TopBP1
is
not
only
essential
for
replication
and
the

replication
checkpoint,
but
is
also
responsible
for
recognizing
sites
of
damage
to

help
launch
a
checkpoint
response.

In
response
to
resected
double
strand
breaks

for
example,
ATM
activation
leads
to
the
phosphorylation
of
many
targets,
one
being

TopBP1.
Direct
ATM
phosphorylation
of
TopBP1
near
its
ATR
activation
domain

(AAD)
at
S1131
in
Xenopus
better
exposes
this
region
to
make
contact
with
ATR,
and

thus
greatly
enhances
ATR’s
kinase
activity
(Yoo,
2007).

It
has
been
proposed
that

TopBP1,
CtBP-‐Interacting
Protein
(CtIP),
and
MRN
form
a
large
complex
that
is

stimulated
by
DNA
damage
to
allow
the
switch
from
ATM
to
ATR
activation
in

response
to
double
strand
breaks
(Ramirez-‐Lugo,
2011).
To
do
this,
ATM
also

controls
resection
of
DNA
ends
by
MRN,
exposing
ssDNA
that
becomes
RPA-‐ssDNA

that
recruits
ATR
to
allow
it
to
launch
a
checkpoint
response
(Yoo,
2009).

In
this

way,
both
resected
double-‐strand
breaks
and
stalled
forks
lead
to
the
generation
of

RPA-‐ssDNA,
which
ultimately
allows
activation
of
ATR
signaling.

1.5
Overview
of
the
projects
presented
in
this
dissertation

The
work
presented
in
this
dissertation
investigates
how
ATR
is
activated
at

stalled
forks
and
resected
double-‐strand
breaks.
Specifically,
I
investigated
how

RPA-‐ssDNA
is
initially
detected
by
TopBP1
to
allow
it
to
serve
in
its
role
in
ATR

activation.
I
find,
that
like
ATR/ATRIP
localization
to
sites
of
damage,
a
direct

interaction
between
TopBP1
and
RPA-‐ssDNA
is
the
mechanism
by
which
TopBP1
is

8

able
to
detect
sites
of
DNA
damage.
This
direct
interaction
between
TopBP1
and

RPA-‐ssDNA
is
mediated
through
its
second
BRCT
domain.
Importantly,
I
also
found

that
BRCT
domains
that
are
similar
to
TopBP1
BRCT2
are
able
to
allow
binding
to

RPA-‐ssDNA
in
other
proteins
as
well.
Furthermore,
I
find
that
ATR
activity
limits
the

amount
of
TopBP1
on
chromatin
after
damage.
Thus,
it
seems
that
a
negative

feedback
loop
of
ATR
activity
limits
further
ATR
activation
once
damage
has
been

sensed.
Lastly,
I
identify
and
characterize
a
TopBP1
mutant
that
allows
the

activation
of
ATR
in
the
absence
of
DNA.

This
ability
is
perhaps
facilitated
by
a

conformational
change
that
allows
the
mutant
to
mirror
a
conformation
TopBP1

normally
undergoes
while
it
is
on
DNA
and
serving
in
its
role
in
ATR
activation.

Altogether,
this
work
provides
a
mechanism
for
how
the
ATR-‐activating
protein,

TopBP1,
recognizes
damage
to
activate
ATR
and
provides
insights
into
how

conformational
changes
in
the
protein
might
allow
it
to
accomplish
this.

9

A

B

C

Figure
1.
Diagram
of
the
various
roles
of
TopBP1
inside
the
cell.
(A)
TopBP1
is

essential
in
the
initiation
step
of
DNA
replication.

It
recognizes
the
cdk-‐2

phosphorylated
Treslin
protein
and
allows
cdc45
loading
onto
origins.

In
yeast,
it

also
is
responsible
for
the
recruitment
of
Sld2
(REQ4L)
protein
as
well.
(B)
TopBP1

loads
onto
undamaged
chromatin
in
a
replication
and
damage-‐independent
manner,

yet
the
significance
of
this
is
not
yet
understood.
(C)
TopBP1
is
essential
for
ATR

activation
in
response
to
replication
stress
and
to
resected
double-‐strand
breaks.

Replication Initiation Unperturbed S phase Replication Stress!
CDT1
CDC6
ORC
MCM 2-7
TOPBP1
TOPBP1
TRESLIN
P
P
TRESLIN
P
P
CDT1
CDC6
ORC
MCM 2-7
CDC 45
RecQ4
TOPBP1 TOPBP1 TOPBP1
911
RPA
TOPBP1
ATR
ATRIP
CHK1
P

10

Chapter
2

Materials
and
Methods

11

Plasmids

All
cloning
was
done
according
to
standard
procedures.

Full-‐length
TopBP1
cDNA

used
was
TopBP1-‐B,
cDNA
encoding
NCBI
accession
number
AAP03894
cloned
into

pCS2+MT.
This
protein
was
initially
named
Xmus101
(see
Van
Hatten
et
al.,
2002).

All
deletion
mutants
were
produced
by
PCR,
using
TopBP1-‐B
cDNA
as
template.

PCR
primers
included
an
NcoI
site
on
the
5’
end
and
an
XhoI
site
on
the
3’end.

Fragments
were
subcloned
into
NcoI/XhoI
digested
pCS2+MT,
and
verified
by
DNA

sequencing.
For
IVT
expression,
TopBP1
deletion
mutants
used
in
Figure
2,
the

amino
acid
sequence
for
each
deletion
mutant
is
given
below:
BRCT0-‐5
(aa
1-‐
758);

BRCT6-‐8
(aa
759-‐
1513);
BRCT0-‐2
(aa
1-‐333);
BRCT3
(aa
333-‐
480);
BRCT4-‐5
(aa

480-‐758);
BRCT0-‐1
(aa
1-‐191);
BRCT1
(aa
99-‐
191);
BRCT1-‐2
(aa
99-‐333);
BRCT2

(aa
191-‐333);
BRCT4
(aa
527-‐622);
BRCT5
(aa
627-‐800);
BRCT4-‐5
(aa
527-‐800);

BRCT4/5
delta
(internal
deletion
of
aa
588-‐760).
Other
vector
plasmid
sequences

correspond
to:
PTIP-‐FL,
amino
acids
1-‐
820
of
the
Xenopus
PTIP
protein,
and
PTIP-‐
BRCT2,
amino
acids
96-‐
186
of
the
Xenopus
PTIP
protein.

All
point
mutants
were

produced
in
the
TopBP1-‐B
cDNA.

Mutations
were
produced
using
a
QuickChangeII

site-‐directed
mutagenesis
kit
(Agilent
Technologies).
For
E.
coli
expression:

GST-‐
BRCT2,
either
wild
type
or
W265R
BRCT2
fragments
described
above
were

subcloned
into
pGEX-‐4T1,
and
His-‐BRCT2,
either
wild
type
or
W265R
BRCT2

fragments
described
above
were
subcloned
into
pET28a.

12

Xenopus
egg
extracts
and
sperm
chromatin
isolation

Egg
extracts
and
sperm
chromatin
were
prepared
as
described
(Yan
et
al.,
2009).

Briefly,
fresh
frog
eggs
were
obtained
and
de-‐jellied
in
2%
cysteine,
spun
at
high

speed
in
centrifuge
and
supplemented
with:
10
ug/ml
aprotinin,
5ug/ml
leupeptin,

3.3ug/ml
nocodazole,
cytochalasin
B,
1
mM
DTT,
and
0.1
mg/ml
cycloheximide.

Prior
to
use,
an
energy
mix
(375
mM
phosphocreatine,
50
mM
ATP,
5
mM
EGTA,
50

mM
MgCl2,
pH
7.4)
was
added
to
egg
extracts
to
supply
ATP
at
1/50
volume.
Sperm

chromatin
was
isolated
from
egg
extract
as
described
(Yan
et
al.,
2009).

Reactions

were
allowed
to
proceed
at
room
temperature
for
the
indicated
time
points,
then

diluted
fourfold
with
NIB
buffer
and
carefully
layered
in
30%
sucrose
in
NIB

cushion.

Tubes
then
washed
three
times
with
NIB
and
chromatin
pellet
was

resuspended
in
2X
Laemmle
sample
buffer
and
boiled.

Expression
vectors
and
IVT
protein
production

All
of
the
expression
vectors
used
for
IVT
protein
production
were
based
on

pCS2+MT.
IVT
reactions
were
performed
using
a
Promega
TnT®
SP6
Quick
Coupled

Transcription/Translation
System,
according
to
the
vendor’s
instructions.

Recombinant
proteins

An
expression
vector
encoding
the
Xenopus
RPA
trimer
was
a
kind
gift
of
K.

Cimprich.

RPA
plasmid
was
expressed
in
BL21
E.
coli
cells
and
purified
following

previously
published
methods.
Insoluble
protein
pellet
was
denatured
in
urea,

13

allowed
to
refold
and
then
purified
on
nickel
agarose
column.
Aliquots
were
then

frozen
liquid
nitrogen
and
stored
at
-‐80C.

GST-‐BRCT2,
GST-‐BRCT2
W265R,
His-‐
BRCT2,
and
His-‐BRCT2
W265R
were
expressed
and
purified
from
E.
coli
using

standard
conditions.

An
expression
vector
encoding
the
Xenopus
Rad
9
tail
domain

was
a
kind
gift
of
K.
Cimprich.

The
protein
was
expressed
and
purified
from
E.
coli

using
standard
conditions.

Replication
assays

Replication
was
measured
by
supplementing
the
replicating
extract
with

radiolabeled
nucleotides,
typically
0.04
uCi/ul
of

∝
32
P-‐dATP
that
are
incorporated

into
the
DNA
through
the
replication
process,
and
treated
with
different
conditions.

These
samples
are
then
collected
at
various
timepoints,
generally
30,
60,
90
and
120

minutes.
Samples
are
then
treated
with
proteinase
K
to
remove
any
associated

proteins
while
leaving
the
DNA
intact
and
then
samples
run
out
on
an
agarose
gel

and
signals
detected
via
a
phospho-‐screen.

ssDNA
binding
assays

Biotin-‐linked
DNA
fragments
of
varying
sizes
were
produced
by
PCR
and
then

denatured
prior
to
coupling
to
Dynabeads®
Streptavidin
(Life
Technologies).

Biotin-‐dsDNA
was
coupled
to
streptavidin
beads
in
BW
buffer
(5mM
Tris,
pH7.4,
1M

NaCl
and
0.5mM
EDTA),

and
treated
with
0.15N
NaOH
to
make
ssDNA.

All
binding

reactions
where
carried
out
in
Buffer
A
(10mM
Hepes,
pH7.6,
80mM
NaCl,
20mM
B-‐

14

glycerol
phosphate,
2.5mM
EGTA
and
0.1%
NP-‐40).

Incubated
for
an
hour
at
room

temperature
for
an
hour,
beads
then
washed
and
eluted
in
2X
Laemmle
sample

buffer.

GST
pull-‐down
assays

Rad9
Pull-‐down:
Target
proteins
were
produced
by
IVT
and
mixed
with
2
ug
GST-‐
Rad9
Tail
and
1
ul
recombinant
casein
kinase
II
(NEB).

Reactions
were
incubated

for
60
minutes
at
30°C
and
then
diluted
into
500
ul
of
Tris-‐buffered
saline
plus
0.1%

Tween-‐20
(TBST).

20
ul
of
washed
glutathione
sepharose
beads
were
then
added

and
incubated
at
4°C
for
one
hour.

Beads
were
washed
three
times
with
TBST
and

then
eluted
with
2XSB.

TopBP1-‐TopBP1
oligomerization:
Myc-‐tagged
and
his-‐tagged
full
length
TopBP1

either
wild
type
or
BRCT4/5
WR
mutant
were
produced
by
IVT
and
then
mixed
and

incubated
for
one
hour
at
room
temperature
to
allow
oligomerization.
Beads

coupled
with
and
without
his-‐antibodies
were
prepared
and
then
these
were

combined
with
binding
buffer
and
reactions
above
for
2hrs
at
4C
with
rotation
for

immunoprecipitation,
then
after
incubation
washed
and
samples
processed
for

Western
blot.

Immunodepletion
and
antibodies

RPA
and
TopBP1
were
depleted
from
Xenopus
egg
extract
as
described
(Yan
et
al.,

2009).

TopBP1
antibodies
have
been
described
(Van
Hatten
et
al.,
2002).

RPA

15

antibodies
were
a
kind
gift
of
J.
Walter.

Monoclonal
antibody
9E10
was
used
to

detect
myc-‐tagged
IVT
proteins,
phosphorylated
Chk1
was
detected
with
Phospho-‐
Chk1
(Ser345)
Antibody
#2341
(Cell
Signaling
Technology),
and
GST
fusion
proteins

were
detected
with
anti-‐GST
antibodies
(Sigma).

16

Chapter
3

Direct
binding
to
RPA-‐coated
ssDNA
allows
recruitment
of
the
checkpoint

activator
TopBP1
to
sites
of
DNA
damage

17

Abstract

A
critical
event
for
the
ability
of
cells
to
tolerate
DNA
damage
and
replication

stress
is
activation
of
the
ATR
kinase.

ATR
activation
is
dependent
on
the
BRCT

repeat-‐containing
protein
TopBP1.

Previous
work
has
shown
that
recruitment
of

TopBP1
to
sites
of
DNA
damage
and
stalled
replication
forks
is
necessary
for

downstream
events
in
ATR
activation,
however
the
mechanism
for
this
recruitment

was
not
known.

Here,
we
use
protein
binding
assays
and
functional
studies
in

Xenopus
egg
extracts
to
show
that
TopBP1
makes
a
direct
interaction,
via
its
BRCT2

domain,
with
RPA-‐coated
ssDNA.

We
identify
a
point
mutant
that
abrogates
this

interaction,
and
show
that
this
mutant
fails
to
accumulate
at
sites
of
DNA
damage,

and
that
the
mutant
cannot
activate
ATR.

These
data
thus
supply
a
mechanism
for

how
the
critical
ATR
activator,
TopBP1,
senses
DNA
damage
and
stalled
replication

forks
to
initiate
assembly
of
checkpoint
signaling
complexes.
Furthermore,
we
find

that
once
TopBP1
has
promoted
initial
ATR
activation,
further
chromatin
loading
of

TopBP1
to
damaged
sites
is
prevented
by
a
negative
feedback
loop
of
ATR
activity.

18

3.1
Introduction

The
maintenance
of
genome
stability
relies
on
faithful
DNA
replication
and

the
ability
of
cells
to
suppress
the
mutagenic
consequences
of
replication
stress
and

DNA
damage.

Two
protein
kinases,
ATM
and
ATR,
are
upstream
of
signaling

cascades
that
control
cell
cycle
progression,
DNA
repair,
replication
fork
stability,

and
transcriptional
responses
to
DNA
damage
and
replication
stress
(reviewed
by

Nam
and
Cortez,
2011;
Maréchal
and
Zou,
2013;
Sirbu
and
Cortez,
2013).

ATM
is

primarily
activated
by
DNA
double-‐strand
breaks
(DSBs),
whereas
stalled

replication
forks
and
DSBs
activate
ATR.

Upon
activation,
ATR
phosphorylates
and

activates
numerous
substrates,
including
the
Chk1
kinase
(Liu
et
al.,
2000;
Guo
et
al,

2000).

Here,
we
address
the
mechanism
for
ATR
activation,
with
a
focus
on
how
the

critical
ATR
activator,
TopBP1,
is
recruited
to
sites
of
DNA
damage.

TopBP1
performs
multiple
functions
in
chromosome
metabolism
(reviewed

by
Wardlaw
et
al.,
2014),
including
the
initiation
of
DNA
replication
(Van
Hatten
et

al.,
2002;
Hashimoto
and
Takisawa,
2003)
and
ATR
activation
(Kumagai
et
al.,

2006).
Previous
work
has
shown
that
the
roles
of
TopBP1
in
replication
initiation

and
ATR
signaling
are
distinct
(Kumagai
et
al.,
2006;
Yan
et
al.,
2006).

Unlike

simpler
eukaryotes
such
as
the
budding
yeast,
where
multiple
factors
including
the

TopBP1
ortholog
Dpb11
can
activate
ATR
(Wanrooij
and
Burgers,
2015),
in

metazoans
TopBP1
is
the
sole
ATR
activator
that
has
been
identified
to
date.

19

Previous
work
has
detailed
important
aspects
of
how
ATR
is
activated
by

stalled
forks.

For
this,
ATR
associates
with
a
binding
partner,
ATR-‐interacting

protein
(ATRIP),
and
the
complex
is
localized
to
stalled
forks
via
direct
interaction

between
ATRIP
and
RPA-‐coated
single-‐stranded
DNA
(RPA-‐ssDNA)
(Cortez
et
al.,

2001;
Zou
et
al.,
2003;
Ball
et
al.,
2005a;
Namiki
et
al.,
2006;
Ball
et
al.,
2005b;
and

Ball
et
al.,
2007).
RPA-‐ssDNA
is
generated
at
stalled
forks
due
to
the
uncoupling
of

DNA
helicase
and
polymerase
activities
that
occurs
when
the
polymerase
stalls
but

the
helicase
does
not
(Byun
et
al.,
2005).

ATRIP-‐mediated
ATR
docking
is
necessary

but
not
sufficient
for
kinase
activation.

Independent
of
ATR-‐ATRIP,
another
protein

complex
forms
on
RPA-‐ssDNA,
and
then
joins
ATR-‐ATRIP
to
activate
ATR
(Zou
et
al,

2002).

This
second
complex
contains,
minimally,
the
Rad9-‐Rad1-‐Hus1
(911)

trimeric
clamp
protein,
the
clamp
loader
Rad17-‐replication
factor
C
(RFC),
and

TopBP1.

Upon
assembly
of
this
second
complex,
TopBP1
is
altered
so
that
an
ATR

activation
domain
is
revealed,
and
this
allows
interaction
between
TopBP1
and

ATR-‐ATRIP
in
a
manner
that
activates
ATR
kinase
(Kumagai
et
al.,
2006
and
Mordes

et
al.,
2008).

TopBP1
and
ATR
also
play
critical
roles
in
the
cellular
response
to
DSBs
in

many
higher
eukaryotes,
including
Drosophila,
Xenopus,
C.
elegans,
and
humans

(Cliby
et
al.,
1998;
Wang
et
al.,
2004;
Garcia-‐Muse
and
Boulton,
2005;
Petersen
et
al.,

2006;
LaRocque
et
al.,
2007;
Kondo
and
Perrimon,
2011).
TopBP1
and
ATR
are
not

only
required
for
the
DSB
response
in
mitotic
cells,
but
also
for
efficient
completion

of
meiotic
recombination
(reviewed
by
Cooper
et
al.,
2014).

The
mechanism
for

20

ATR
activation
at
sites
of
DSBs
is
not
fully
understood.

Work
in
Xenopus
has

outlined
a
pathway
whereby
DSBs
activate
ATM,
which
then
phosphorylates

TopBP1,
and
this
allows
TopBP1
to
activate
ATR
(Yoo
et
al.,
2007).

Previous
work

has
also
shown
that
in
human
cells
containing
DSBs,
ATR
activity
towards
different

substrates
requires
distinct
activation
modes.

While
TopBP1
is
required
for
ATR

activity
in
all
cases,
Rad17
(and
by
extension
the
911
complex)
is
more
important

for
Chk1
phosphorylation
while
the
Nbs1
protein
is
more
important
for
ATR-‐
directed
phosphorylation
of
RPA32
(Shiotani
et
al.,
2013).

A
dual
role
of
Rad17
and

Nbs1
in
ATR
activation
has
also
been
observed
in
Xenopus
(Yoo
et
al.,
2009;
Lee
and

Dunphy,
2013).

While
much
remains
to
be
learned
about
how
ATR
is
activated
at

DSBs,
what
is
clear
is
that
TopBP1
is
essential
to
this
critical
process.

Given
the
central
role
of
TopBP1
in
ATR
signaling
at
both
stalled
forks
and

DSBs
it
is
important
to
understand
how
TopBP1
is
recruited
to
these
different
DNA

structures.

At
stalled
forks,
TopBP1
can
form
a
complex
with
911-‐Rad17-‐RFC
by

virtue
of
an
interaction
between
the
BRCT
1
and
2
domains
within
TopBP1
and
the

C-‐terminal
tail
domain
of
Rad9
(Delacroix
et
al.,
2007;
Lee
et
al.,
2007;
Lee
et
al.,

2010;
and
Rappas
et
al.,
2011).

This
interaction
requires
phosphorylation
of
Rad9

on
S373
(in
the
Xenopus
protein),
a
site
which
is
thought
to
be
constitutively

phosphorylated
by
casein
kinase
II
(Takeishi
et
al.,
2010).

The
TopBP1-‐Rad9
tail

interaction
is
required
for
the
ability
of
TopBP1
to
contact
DNA-‐bound
ATR-‐ATRIP

complexes,
and
for
ATR
activation
(Delacroix
et
al.,
2007
and
Lee
et
al.,
2007).

The

discovery
of
the
TopBP1-‐Rad9
interaction
led
to
a
simple
model
for
TopBP1

21

recruitment,
whereby
the
911
clamp
is
loaded
by
Rad17-‐RFC,
and
TopBP1
is
then

recruited
via
interaction
with
the
Rad9
tail.

Four
recent
findings,
however,
have

challenged
this
view,
and
suggest
that
initial
TopBP1
recruitment
to
sites
of
damage

is
independent
of
911.

First,
multiple
lines
of
published
evidence
point
to
a
direct
role
for
TopBP1
in

911
loading.

A
study
from
our
laboratory
has
shown
that
when
replicating

chromatin
is
isolated
and
transferred
to
Xenopus
egg
extract
lacking
TopBP1,
911

fails
to
load
onto
stalled
replication
forks
(Yan
et
al.,
2009).

Furthermore,
chromatin

transfer
experiments
showed
that
TopBP1
must
be
physically
present
with
911
for

911
to
load
onto
stalled
forks.

In
addition,
a
TopBP1
point
mutant
(W265R)
was

identified
that
cannot
accumulate
at
sites
of
replication
stress,
and,
in
egg
extract

containing
this
mutant
as
the
sole
source
of
TopBP1,
911
fails
to
load
onto
stalled

replication
forks
and
ATR
is
not
activated
(Yan
et
al.,
2009).

And
finally,
in
human

cells,
siRNA-‐mediated
depletion
of
TopBP1
prevents
recruitment
of
911
to

chromatin
after
hydroxyurea
treatment
(Gong
et
al.,
2010)
or
UV
irradiation
(Ohashi

et
al.,
2014).

These
data
strongly
suggest
that
TopBP1
recruits
911,
and
not
the

other
way
around.

Second,
it
has
been
demonstrated
that
the
TopBP1-‐Rad9
tail
interaction
is

dispensable
for
initial
recruitment
of
TopBP1
to
stalled
forks
(Lee
et
al.,
2010).

In

these
studies,
TopBP1
was
shown
to
accumulate
on
stalled
forks
even
when
Rad9

tail
phosphorylation
was
prevented
by
a
S373A
mutation.

Third,
in
the
context
of

22

meiotic
DSBs
in
mice,
TopBP1
recruitment
is
unhindered
in
Hus1
conditional
knock-‐
out
spermatocytes
(Lyndaker
et
al.,
2013).

Fourth,
for
ATR
activity
towards
RPA32

at
DSBs
in
human
cells,
Rad17
is
dispensable
but
TopBP1
is
not
(Shiotani
et
al.,

2013).

These
recent
findings
call
for
a
revision
to
the
simple
model
where
TopBP1
is

recruited
to
sites
of
damage
by
virtue
of
the
interaction
with
the
Rad9
tail.

Rather,

TopBP1
accumulates
at
stalled
forks
and
DSBs
via
a
previously
unknown

mechanism,
and
once
it
is
there
then
911
loading
and
ATR
activation
follow.

In

order
to
fully
understand
how
ATR
is
activated
during
replication
stress
and
DNA

damage,
it
is
therefore
necessary
to
understand
the
mechanism
for
the
initial

TopBP1
recruitment
to
stalled
forks
and
DSBs.

As
detailed
below,
we
have

elucidated
this
mechanism,
and
it
involves
direct
interaction
between
the
TopBP1

BRCT2
domain
and
RPA-‐ssDNA.

23

3.2
Results

TopBP1
binds
ssDNA
in
a
length-‐
and
RPA-‐dependent
manner

Previous
studies
have
revealed
that
the
platform
for
ATR
signaling
is
the

RPA-‐ssDNA
that
becomes
exposed
upon
uncoupling
of
helicase
and
polymerase

activities
during

replication
stress,
or
after
resection
of
DSBs
(reviewed
by

Marechal
et
al,
2015).

Given
that
RPA-‐ssDNA
is
a
common
feature
of
both
stalled

forks
and
DSBs,
and
that
TopBP1
activates
ATR
in
both
contexts,
we
reasoned
that

interaction
between
TopBP1
and
RPA-‐ssDNA
is
likely
to
be
important
for
how

TopBP1
is
recruited
to
sites
of
damage.

We
therefore
examined
the
ability
of

TopBP1
to
bind
to
ssDNA
after
incubation
in
Xenopus
egg
extract.

Magnetic

streptavidin
beads
were
coupled
to
either
buffer
or
equal
amounts
of
either
dsDNA

or
ssDNA
and
then
incubated
in
egg
extract
(Fig.
2A).

Following
incubation,
the

beads
were
isolated,
washed,
and
protein
binding
was
assessed
by
Western
blot.

As

shown
in
Fig.
2A,
TopBP1
could
bind
well
to
ssDNA
beads,
but
did
not
efficiently

bind
either
dsDNA
beads
or
beads
alone.

To
better
characterize
TopBP1’s
binding

to
ssDNA,
we
next
determined
size
requirements
for
binding.

We
produced
ssDNAs

of
different
lengths
and
these
were
incubated
in
egg
extract,
recovered,
washed,
and

assayed
for
protein
binding.

As
shown
in
Fig.
2B,
efficient
binding
of
TopBP1
was

observed
for
ssDNAs
of
>250
nt;
however,
binding
was
not
observed
for
a
100
nt

piece
of
ssDNA.

Interestingly,
RPA
could
bind
efficiently
to
the
100
nt
ssDNA.

These

data
reveal
a
size
threshold
for
efficient
TopBP1
binding
to
the
ssDNA,
between
100

and
250
nt
(Fig.
2B;
Experiment
performed
by
Shan
Yan).

24

To
pursue
these
observations,
we
next
asked
if
RPA
is
required
for
TopBP1

interaction
with
ssDNA
in
egg
extract.

When
RPA
was
removed
from
the
extract
by

immunodepletion,
TopBP1
could
not
bind
efficiently
to
ssDNA
beads,
although

binding
was
observed
in
the
mock-‐depleted
control
extract
(Fig.
2C;
Experiment

performed
by
Shan
Yan).

This
suggests
that
RPA
is
necessary
for
TopBP1
to
bind

ssDNA
in
egg
extract.

To
ask
if
RPA
is
sufficient
for
TopBP1
ssDNA
binding,
we

prepared
a
recombinant,
purified
form
of
the
RPA
trimer,
and
used
it
to
prepare

RPA-‐ssDNA
templates
for
TopBP1
binding
assays.

Recombinant,
myc-‐tagged

TopBP1
was
produced
by
in
vitro
transcription
and
translation
(IVT)
in
rabbit

reticulocyte
lysates.

IVT
TopBP1
was
then
incubated
with
purified
RPA
and
ssDNA

and
assayed
for
binding.

We
observed
that
TopBP1
bound,
in
an
RPA
dose-‐
dependent
manner,
to
the
ssDNA
beads
(Fig.
2D).

Binding
was
not
observed
when

RPA
was
omitted
from
the
reactions.

We
next
asked
if
TopBP1
could
bind
to
the

RPA
trimer
in
the
absence
of
ssDNA.

RPA
trimer
was
immobilized
on
nickel
(NTA)-‐
agarose
beads
by
virtue
of
a
6-‐histidine
tag
on
RPA70,
and
incubated
with
IVT

TopBP1.

As
shown
in
Fig.
2E,
TopBP1
did
not
bind
to
free
RPA.

Based
on
these
data,

we
conclude
that
TopBP1
binds
to
RPA-‐ssDNA,
but
not
ssDNA
alone
or
RPA
trimer

alone.

We
next
asked
if
there
was
any
difference
for
TopBP1
binding
between
RPA-‐
ssDNA
formed
in
egg
extract
versus
RPA-‐ssDNA
formed
with
purified,
recombinant

RPA.

For
this,
ssDNA
beads
were
incubated
either
with
egg
extract
or
purified
RPA.

RPA-‐ssDNA
complexes
were
then
isolated,
washed,
and
incubated
with
IVT
TopBP1.

As
shown
in
Fig.
2F,
recombinant
RPA
was
just
as
good
as
the
RPA
in
egg
extract
in

allowing
TopBP1
binding
to
the
ssDNA
beads.

This
shows
that
any
post-‐

25

translational
modifications
on
RPA
that
occur
in
egg
extract
are
dispensable
for

TopBP1
binding.

TopBP1
uses
its
BRCT2
domain
to
bind
RPA-‐ssDNA

To
further
analyze
the
TopBP1-‐RPA-‐ssDNA
interaction
identified
in
Fig.
2,
we

used
deletion
analysis
to
elucidate
the
TopBP1
sequence
determinants
required
for

binding.

TopBP1
is
composed
of
9
copies
of
the
BRCT
domain,
termed
0
through
8

(Rappas
et
al.,
2011),
that
are
scattered
throughout
the
length
of
the
protein
(Fig.

3A).

For
our
domain
delineation
experiments,
myc-‐tagged
fragments
of
TopBP1

were
produced
by
IVT
and
then
incubated
with
ssDNA
and,
optionally,
purified

recombinant
RPA.

Binding
was
then
assessed
by
Western
blot.

Bifurcation
of
the

protein
into
N-‐terminal
and
C-‐terminal
portions
revealed
that
the
N-‐terminal
half,

containing
BRCTs
0-‐5,
bound
ssDNA
in
an
RPA-‐dependent
manner,
whereas
the
C-‐
terminal
half
did
not
(Fig.
3B).

Subdivision
of
the
N-‐terminal
half
revealed
that

fragments
containing
BRCTs
0-‐2
and
4-‐5
bound
ssDNA
in
an
RPA-‐dependent

manner,
whereas
a
fragment
containing
BRCT3
did
not
(Fig.
3C).

Further
analysis
of

the
region
containing
BRCTs
0-‐2
revealed
that
BRCT2
alone
can
bind
efficiently
to

ssDNA,
in
an
RPA-‐dependent
manner,
whereas
fragments
containing
either
BRCTs

0-‐1
or
BRCT1
alone
could
not
(Figs.
3D&E).

These
data
reveal
that
BRCT2
binds

well
to
RPA-‐ssDNA,
and
that
some
binding
is
also
observed
with
the
BRCT4-‐5

fragment.

To
assess
the
importance
of
the
BRCT4-‐5
domains
for
binding
of
TopBP1

to
RPA-‐ssDNA,
we
tested
an
internal
deletion
mutant
that
lacks
these
sequences

(TopBP1
Δ4&5).

As
shown
in
Fig.
3F,
this
protein
bound
as
well
as
its
wild
type

26

counterpart
to
RPA-‐ssDNA.

We
therefore
focused
on
the
interaction
between

BRCT2
and
RPA-‐ssDNA
for
the
remainder
of
this
study.

Having
identified
TopBP1
BRCT2
as
an
RPA-‐ssDNA
binding
domain,
we

searched
for
other
proteins
that
contain
similar
BRCT
domains.

This
identified
the

second
BRCT
domain
within
the
DSB-‐response
protein
PTIP
as
a
candidate
RPA-‐
ssDNA
binding
domain
(Figs
4A-‐B).

We
observed
that
the
BRCT2
domain
from
PTIP

bound
ssDNA
in
an
RPA-‐dependent
manner,
and
that
binding
required
that
the

ssDNA
be
larger
than
100
nt
(Fig.
4C),
as
is
the
case
for
TopBP1
BRCT2
(Fig.
3E).

Thus,
binding
to
RPA-‐ssDNA
is
a
general
feature
for
a
subclass
of
BRCT
domains.

Our
finding
that
TopBP1
BRCT2
is
an
RPA-‐ssDNA
binding
domain
was

intriguing
given
previous
work
from
our
laboratory
showing
that
a
single
point

mutation
within
BRCT2,
W265R,
prevents
TopBP1
from
accumulating
at
stalled

replication
forks,
but
nonetheless
supports
replication
initiation
(Yan
et
al,
2009).

To
pursue
this
connection,
we
next
asked
if
TopBP1
W265R
could
bind
RPA-‐ssDNA

using
the
assays
employed
here.

IVT
wild
type
and
W265R
TopBP1
proteins
were

mixed
with
egg
extract
and
either
no
DNA,
dsDNA,
or
ssDNA,
and
binding
was

assessed
as
in
Fig.
2A.

As
expected,
wild
type
TopBP1
bound
to
ssDNA,
however
the

W265R
mutant
did
not
show
detectable
binding
(Fig
5A).

Furthermore,
when
the

same
IVT
TopBP1
proteins
were
mixed
with
ssDNA
and
purified
RPA,
we
again

observed
that
wild
type,
but
not
W265R
TopBP1,
could
bind
ssDNA
efficiently,
in
an

RPA-‐dependent
manner
(Fig.
5B).

We
next
asked
if
the
W265R
mutation
in
the

context
of
BRCT2
alone
would
impact
binding
to
RPA-‐ssDNA.

For
this
experiment,

purified
recombinant
forms
of
GST-‐tagged
BRCT2
either
wild
type
or
containing
the

27

W265R
mutation
were
prepared
and
used
for
binding
assays.

As
shown
in
Fig.
5C,

GST-‐BRCT2
bound
well
to
ssDNA
in
an
RPA-‐dependent
manner,
however
GST-‐
BRCT2
W265R
did
not.

This
experiment
makes
two
important
points.

First,

because
this
was
a
completely
purified
system,
we
see
that
BRCT2
can
bind
directly

to
RPA-‐ssDNA,
and
furthermore,
because
all
proteins
were
purified
from
E.
coli,
no

post-‐translational
modifications
on
either
BRCT2
or
RPA
are
required
for
binding.

Second,
the
data
show
that
W265
is
a
critical
determinant
for
binding
to
RPA-‐
ssDNA,
both
in
the
context
of
the
full-‐length
protein
(Fig.
5A-‐B)
and
for
BRCT2
alone

(Fig.
5C).

We
next
asked
if
the
ssDNA
size
restrictions
observed
previously
for
binding

of
full-‐length
TopBP1
(Fig.
2B)
also
applied
to
the
isolated
BRCT2
domain,
and

found
this
was
indeed
the
case.

IVT-‐produced
BRCT2
bound
to
RPA-‐ssDNA
of
150

nt,
but
did
not
bind
when
the
ssDNA
was
smaller
than
150
nt
(Fig.
5D).

Based
on
the

ability
of
the
isolated
BRCT2
domain
to
compete
with
the
full-‐length
protein
for

ssDNA
binding,
and
the
similarity
in
ssDNA
size
restrictions
for
binding
in
both
full-‐
length
protein
and
BRCT2
domain,
we
conclude
that
the
isolated
BRCT2
domain

binds
to
RPA-‐ssDNA
in
a
similar
manner
as
the
full-‐length
protein.

Previous
work
has
shown
that
the
tandem
BRCT1-‐2
repeats
of
TopBP1
bind

to
the
phosphorylated
Rad9
tail
domain
(Lee
et
al,
2007;
Rappas
et
al,
2011),
and
we

have
shown
here
that
BRCT2
alone
binds
to
RPA-‐ssDNA
(summarized
in
Fig.
5E).

The
amino-‐terminal
region
of
the
TopBP1
protein
thus
appears
to
interact
with

28

multiple
binding
partners
that
are
important
for
checkpoint
signaling.

Although

previous
work
had
narrowed
down
the
Rad9
binding
region
of
TopBP1
to
BRCT1-‐2

(Rappas
et
al,
2011),
to
our
knowledge
the
ability
of
either
isolated
BRCT1
or
BRCT2

to
bind
the
Rad9
tail
domain
has
not
yet
been
tested.

This
was
of
interest,
as
we

wanted
to
know
if
the
binding
determinants
within
the
BRCT1-‐2
region
for
Rad9

relative
to
RPA-‐ssDNA
were
identical,
or
distinct
despite
being
located
in
the
same

region
of
the
protein.

To
examine
this,
we
performed
a
pull-‐down
assay
with
a

previously
described
GST-‐Rad9
tail
domain
fusion
protein
(Duursma
et
al,
2013)

and
looked
for
interaction
between
the
Rad9
tail
domain
and
TopBP1
BRCT0-‐2,

BRCT1,
or
BRCT2.

As
shown
in
Fig.
3E,
the
BRCT0-‐2
fragment
could
bind
well
to
the

Rad9
tail
domain,
as
expected,
however
neither
BRCT1
or
BRCT2
alone
could
bind.

It
thus
appears
that
the
tandem
BRCT1-‐2
repeats
bind
Rad9,
whereas
BRCT2
alone

is
sufficient
to
bind
RPA-‐ssDNA
(Fig.
3E;
Experiment
performed
by
Matt
Michael).

We
conclude
that
distinct
determinants
control
binding
of
TopBP1
to
Rad9
and

RPA-‐ssDNA.

TopBP1
BRCT2
W265R
fails
to
accumulate
on
DNA
DSB-‐containing
chromatin

and
fails
to
activate
ATR
during
a
DSB
response

Previous
work
has
shown
that
TopBP1
W265R
is
not
recruited
efficiently
to

stalled
replication
forks
(Yan
et
al,
2009),
and
data
shown
here
demonstrate
that

this
mutant
also
fails
to
bind
efficiently
to
RPA-‐ssDNA
(Figs.
5A-‐C;
Experiment
5C

performed
by
Shan
Yan).

Taken
together,
the
data
strongly
suggest
that
a
BRCT2-‐

29

mediated
direct
interaction
with
RPA-‐ssDNA
allows
TopBP1
to
accumulate
on
the

RPA-‐ssDNA
present
at
stalled
forks.

To
see
if
this
a
general
mechanism
for
how

TopBP1
is
recruited
to
sites
of
DNA
damage,
we
next
assessed
the
ability
of
TopBP1

W265R
to
accumulate
on
chromatin
during
a
DSB
response,
given
that
DSB-‐
containing
chromatin
also
contains
RPA-‐ssDNA.

Egg
extracts
were
prepared,

immunodepleted
of
endogenous
TopBP1,
and
optionally
supplemented
with
either

blank
IVT,
or
IVTs
expressing
wild
type
or
W265R
TopBP1
(Fig.
5F).

These
extracts

were
then
mixed
with
sperm
chromatin
and
the
EcoRI
restriction
enzyme,
which

activates
ATR
by
inducing
DSBs
(Yoo
et
al,
2006).

After
incubation,
the
chromatin

was
isolated
and
probed
for
myc-‐tagged
TopBP1
proteins
and,
as
a
loading
control,

the
Orc2
protein.

In
addition,
samples
of
the
total
extract
were
taken
to
blot
for

phosphorylated
Chk1
(Chk1-‐P),
as
a
readout
for
ATR
activity.

As
shown
in
Fig.
5G,

wild
type
TopBP1
was
bound
to
the
DSB-‐containing
chromatin,
however
TopBP1

W265R
did
not
bind
to
DSB-‐containing
chromatin.

Furthermore,
while
wild
type

TopBP1
could
support
Chk1
phosphorylation
during
the
DSB
response,
TopBP1

W265R
could
not.

These
data
are
consistent
with
our
previous
work
that
revealed

defects
for
TopBP1
W265R
in
associating
with
stalled
forks,
and
suggest
that
direct

binding
to
RPA-‐ssDNA
is
a
general
mechanism
that
allows
TopBP1
to
be
recruited
to

sites
of
damage
to
promote
ATR
activation.

ATR
regulates
TopBP1
association
with
chromatin

In
a
final
experiment,
we
asked
if
checkpoint
kinase
activity
could
influence

TopBP1
recruitment
to
chromatin
during
a
DNA
damage
response.

Sperm

30

chromatin
was
added
to
egg
extract
and
the
samples
were
optionally
treated
with

chemical
inhibitors
of
either
ATM
or
ATR
(ATMi
and
ATRi,
respectively)
and
EcoRI

to
produce
DSBs.

After
incubation
the
chromatin
was
isolated
and
probed
for

TopBP1
and
Orc2,
as
a
loading
control.

As
shown
in
Fig.
16
lanes
1
and
2,
the

presence
of
DSBs
caused
increased
levels
of
TopBP1
on
chromatin,
as
expected.

Addition
of
ATMi
did
not
affect
the
amount
of
TopBP1
on
DSB-‐containing
chromatin

(lane
3),
however
ATRi
had
a
dramatic
effect,
as
TopBP1
levels
were
substantially

increased
(lane
4).

ATRi
also
increased
the
amount
of
TopBP1
on
undamaged

chromatin
(lane
5),
however
these
levels
were
reduced
relative
to
the
sample

containing
DSBs
and
ATRi.

We
note
that
a
high
concentration
of
sperm
chromatin

(4,000/ul)
was
used
for
this
experiment,
and
previous
work
has
shown
that
ATR
is

quite
active
at
this
concentration,
even
without
DNA
damage
(Murphy
and
Michael,

2013).

These
data
suggest
the
possibility
of
a
negative
feedback
loop,
whereby
once

ATR
is
activated
it
can
suppress
the
further
recruitment
of
TopBP1,
and
thereby

limit
the
number
of
checkpoint
signaling
complexes
that
assemble
on
DSBs.
Exactly

how
ATR
activity
is
limiting
TopBP1
chromatin
association
is
not
clear,
but
it
is

tempting
to
speculate
that
direct
inhibition
of
TopBP1
chromatin
association
by

ATR
is
the
reason.
When
DSB’s
in
the
form
of
linearized
plasmid
DNA
is
added
to

extract,
supplemented
optionally
with
inhibitors
against
ATM,
ATR,
or
both,
we
see

a
damage
induced
phosphorylation
shift
is
lost,
most
notably
by
the
inhibition
of

ATR
activity
(Figure
6B).
Thus,
it
is
possible
that
ATR
directly
limits
TopBP1

chromatin
association
through
phosphorylation.

31

3.3
Discussion

Here
we
sought
to
determine
the
mechanism
by
which
TopBP1
senses
DNA

damage
to
activate
the
checkpoint.

We
find
that
TopBP1
is
able
to
bind
RPA-‐ssDNA

directly
through
its
BRCT2
domain
to
activate
the
checkpoint,
both
to
stalled
forks

and
sites
of
double
strand
breaks.
We
found
that
there
is
a
size
requirement
of
100-‐
250
nucleotides
for
TopBP1
RPA-‐ssDNA
binding,
which
corresponds
that
at
least
3-‐
5
RPA
molecules
on
the
DNA.
We
find
that
RPA-‐ssDNA
is
required,
as
TopBP1
does

not
bind
either
DNA
or
RPA
when
alone
and
not
in
the
context
of
RPA-‐ssDNA.

Additionally,
we
find
that
no
post-‐translational
modification
on
RPA
is
required
for

the
interaction.

We
further
showed
that
BRCT2
is
the
main
binding
domain
to
RPA-‐
ssDNA
and
that
this
interaction
is
direct.

We
show
that
RPA-‐ssDNA
binding
is
a

conserved
feature
of
a
subset
of
BRCT
domains.
We
also
find
that
W265
within

BRCT2
of
TopBP1
is
a
critical
determinant
for
RPA-‐ssDNA
binding,
as
mutating
this

residue
prevents
both
RPA
binding
and
checkpoint
activation,
revealing
that
direct

association
between
TopBP1
and
RPA-‐ssDNA
is
required
to
activate
ATR.

Once
ATR

activity
is
high,
further
loading
of
TopBP1
to
sites
of
damage
decreases,
suggesting
a

negative
feedback
loop
of
ATR
activity
that
limits
TopBP1
chromatin
association

once
damage
has
been
detected.

Data
shown
here
reveal
new
insights
into
how
DSBs
and
stalled
replication

forks
are
sensed
by
the
ATR
pathway
and
how
ATR
is
activated
in
response
to
this

damage.

In
Fig.
7
we
present
a
new
model
for
ATR
activation
at
stalled
forks.

This

model
proposes
that
an
early
event
in
ATR
activation
is
the
direct
interaction

32

between
TopBP1
and
RPA-‐ssDNA
at
the
stalled
fork.

TopBP1
binding
then
allows

recruitment
of
Rad17-‐911,
perhaps
through
an
interaction
that
changes
the

conformation
of
Rad17-‐911,
thereby
allowing
it
to
more
productively
interact
with

the
5’-‐DNA
junction
(as
depicted
in
the
figure),
or
through
clearance
of
other
factors

that
might
preclude
interaction
between
Rad17-‐911
and
the
5’-‐DNA
junction.

Clamp
loading
then
ensues,
and
we
propose
that
this
event
stabilizes
TopBP1
at
the

stalled
fork,
possibly
because
TopBP1
moves
from
its
binding
site
on
RPA-‐ssDNA
to

a
higher-‐affinity
binding
site
on
the
Rad9
tail
domain
(as
depicted
in
the
figure),
or

because
additional
molecules
of
TopBP1
join
the
complex
via
binding
to
Rad9.

Stabilization
of
TopBP1
at
the
stalled
fork
after
clamp
loading
is
consistent
with

previous
work
showing
that
TopBP1
recruitment
to
stalled
forks
is
reduced
(but
not

eliminated)
upon
Rad17
depletion
(Lee
et
al.,
2010).

Finally,
once
TopBP1
is
bound

to
Rad9,
it
can
interact
with
the
independently
recruited
ATR-‐ATRIP
complex,
and

ATR
is
activated.

In
summary,
our
new
data,
combined
with
previous
results,
show

that
two
sensors
independently
recognize
stalled
forks,
ATRIP
and
TopBP1,
and
that

a
common
mechanism
is
employed:
direct
interaction
between
these
factors
and

RPA-‐ssDNA.

These
findings
underscore
the
importance
of
RPA-‐ssDNA
as
a
platform

for
checkpoint
complex
assembly
and
activation.

33

Figure
2.

TopBP1
binds
ssDNA
in
an
RPA-‐
and
length-‐dependent
manner.

(A)
The
depicted
biotin-‐coupled
DNAs
were
mixed
with
50
ul
of
Xenopus
egg

extract,
isolated
using
streptavidin
magnetic
beads,
washed,
eluted,
and
bound

proteins
detected
by
Western
blot.

Blots
were
probed
with
the
indicated
antibodies.

XEE
refers
to
1
ul
of
egg
extract.
(B)
Same
as
(A)
except
the
size
of
ssDNA
was

varied,
as
depicted.
(C)
Same
as
(A)
except
Xenopus
egg
extract
was
either
mock-‐
depleted
or
depleted
of
RPA
using
anti-‐RPA
antibodies,
as
indicated.
(D)
IVT
myc-‐
tagged
TopBP1
was
mixed
with
varying
amounts
of
purified,
recombinant
RPA
and

biotin-‐coupled
ssDNA
(1.5
kb).

The
ssDNAs
were
isolated
using
streptavidin

magnetic
beads,
washed,
eluted,
and
bound
proteins
detected
by
Western
blot.

TopBP1
was
detected
using
Mab
9E10,
which
recognizes
the
myc
tag,
and
RPA
was

detected
using
antibodies
against
the
Xenopus
RPA
trimer.
(E)
Purified,

recombinant
RPA
trimer
was
optionally
coupled
to
nickel
NTA
agarose
beads
by

virtue
of
a
6-‐histidine
tag
on
the
70
kDa
subunit.

Beads
were
mixed
with
IVT
myc-‐
tagged
TopBP1,
washed,
and
bound
proteins
detected
by
Western
blot.

TopBP1
and

RPA
were
detected
as
in
(D).

Input
refers
to
5%
of
the
initial
reaction
volume.
(F)

Biotin-‐linked
ssDNA
was
mixed
with
either
egg
extract
(XEE)
or
the
indicated

amount
of
purified
RPA.

The
ssDNAs
were
isolated
using
streptavidin
magnetic

beads,
washed,
and
then
incubated
with
IVT
myc-‐tagged
TopBP1.

The
beads
were

isolated
again,
washed,
and
bound
proteins
detected
as
in
(D).

34

Figure 1!
biotin!
no DNA!
dsDNA (1.5 kbp)!
ssDNA (1.5 kb)!
A!
TopBP1!
RPA70!
ssDNA (kb)!
3! 1! .5! .25! .1! 0!
B!
TopBP1!
RPA70!
TopBP1!
RPA70!
RPA32!
RPA-!
depleted!
mock-!
depleted!
C!
D!
myc-!
TopBP1!
no RPA!
RPA!
RPA70!
+ ssDNA!
input!
RPA70!
RPA!
beads!
empty!
beads!
E!
myc-!
TopBP1!
RPA70!
XEE!
10! 20!
rRPA (ul)!
ssDNA beads + XEE or rRPA!
!
recover ssDNA & wash!
!
+ IVT TopBP1-B!
!
wash & elute bound proteins!
F!
myc-!
TopBP1!

35

Figure
3.

Deletion
analysis
of
TopBP1
binding
to
RPA-‐ssDNA.
(A)
Cartoon

depicting
the
relative
positions
of
BRCT
domains
(numbered)
within
the
TopBP1

protein.
(B-‐F)
Myc-‐tagged
TopBP1
dervitives
corresponding
to
the
indicated
BRCT

domain(s)
were
produced
by
IVT
and
then
mixed
with
biotin-‐linked
ssDNA
(1.5
kb)

and
binding
buffer.

Recombinant
RPA
was
optionally
added,
as
indicated.

After

incubation,
the
ssDNAs
were
isolated
using
streptavidin
magnetic
beads,
washed,

eluted,
and
bound
proteins
detected
by
Western
blot.

The
TopBP1
fragments
were

detected
using
Mab
9E10,
which
recognizes
the
myc
tag.

Input
refers
to
1.6%
of
the

initial
binding
reaction.

36

Figure 2!
TopBP1!
0! 1! 2! 3! 4! 5! 6! 7! 8!
A!
0-5 !
C!
- +!
+ ssDNA!
RPA:!
BOUND!
INPUT!
0-2!
- +!
3!
- +!
4-5 !
- +!
BRCT:!
BOUND!
INPUT!
0-2 !
E!
- +!
+ ssDNA!
RPA:!
1-2!
- +!
2!
- +!
BRCT:!
0-2 !
D!
- +!
+ ssDNA!
RPA:!
0-1!
- +!
1!
- +!
BRCT:!
BOUND!
INPUT!
BOUND!
INPUT!
0-5 !
- +!
+ ssDNA!
RPA:!
6-8!
- +!
BRCT:!
B!
- +! RPA:! - +!
WT!
BOUND!
TopBP1 + ssDNA!
INPUT!
!4&5!
F!

37

Figure
4.
PTIP
BRCT2
binds
ssDNA
in
an
RPA-‐
and
length-‐dependent
manner.

(A)
Cartoons
depicting
the
TopBP1
and
PTIP
proteins
with
BRCT
domains
boxed

and
numbered.
(B)
Amino
acid
sequence
alignment
for
the
indicated
BRCT
domains.

The
protein
secondary
structure
signatures
for
b
pleated
sheets
(b)
and
a
helices
(a)

are
shown
above
the
alignment.
(C)
RPA-‐ssDNA
binding
assays
with
the
indicated

proteins
were
performed
exactly
as
described
for
Figs
3B-‐F.

TopBP1 BRCT2!IFRGCTICVTGLSSLDRKEVQRLTALHGGEYTGQLKMN-ESTHLIV!
PTIP BRCT2 !IFFGVTACLSQVSPDDRNSLWALTTFYGGDC--QLSLNKKCTHLIV!
TopBP1 BRCT3 !LLDGCRIYLCGFGGRKLDKLRKLINNGGGVRFNQLTGDV--THIIV!
TopBP1 BRCT2!QEAKGQKYECAR-KWIVHC-ISVQWFFDSIEKGFCQDETMYKI !
PTIP BRCT2 !PEPKGNKYEYAFQRGSIKI-VTPDWVLDSVSEKTKKDEALYHP!
TopBP1 BRCT3 !GETDEELKQFLNKTQHRPYVLTVKWLLDSFAKGHLQPEEIYFH !
!1! "1! !2! !3!
"2! "3! !4!
B!
TopBP1!
0! 1! 2! 3! 4! 5! 6! 7! 8!
1! 2! 3! 4! 5! 6!
PTIP!
A!
C!
- + - + - + - +!
TopBP1 TopBP1 PTIP PTIP!
BRCT2 BRCT3 BRCT2 BRCT2!
1.5KB 1.5KB 1.5KB 100bp!
BOUND!
INPUT!
BRCT:!
RPA:!
+ ssDNA!

38

Figure
5.

Functional
characterization
of
the
TopBP1
BRCT2
interaction
with

RPA-‐ssDNA.
(A)
Myc-‐tagged
full-‐length
TopBP1
proteins,
either
wild
type
or
the

W265R
point
mutant,
were
produced
by
IVT
and
then
mixed
with
egg
extract
and

the
indicated
DNAs.

Samples
were
then
processed
exactly
as
in
Fig.
2A.

Proteins

were
detected
with
Mab
9E10
against
the
myc
tag.
(B)
Myc-‐tagged
full-‐length

TopBP1
proteins,
either
wild
type
or
the
W265R
point
mutant,
were
produced
by

IVT
and
then
utilized
for
the
ssDNA
binding
assay,
as
described
in
Figs.
3B-‐F.
(C)

Purified,
recombinant
purified
GST-‐BRCT2
fusion
proteins,
either
wild
type
or
the

W265R
mutant,
were
mixed
with
biotin-‐linked
ssDNA
(1.5
kb)
and
binding
buffer.

Recombinant
RPA
was
optionally
added,
as
indicated.

After
incubation,
the
ssDNAs

were
isolated
using
streptavidin
magnetic
beads,
washed,
eluted,
and
bound

proteins
detected
by
Western
blot
using
anti-‐GST
antibodies.

Input
refers
to
1.6%
of

the
initial
binding
reaction.
(D)
Myc-‐tagged
TopBP1
BRCT2
fragment
was
produced

by
IVT
and
then
mixed
with
recombinant
RPA
and
biotin-‐linked
ssDNAs
of
the

indicated
size.

The
samples
were
then
processed
as
in
Figs.
3
B-‐F.

Input
refers
to

1.6%
of
the
initial
binding
reaction.
(E)
Cartoon
depicting
the
N-‐terminal
region
of

TopBP1
and
binding
determinants
for
Rad9
and
RPA-‐ssDNA.

Myc-‐tagged
TopBP1

fragments,
corresponding
to
the
indicated
BRCT
domains,
were
produced
by
IVT

and
then
mixed
with
purified
Xenopus
Rad9
tail
domain
fused
to
GST
(GST-‐Rad9-‐T).

Recombinant
casein
kinase
2
was
also
included
in
the
reaction.

After
incubation,

proteins
bound
to
GST-‐Rad9-‐T
were
recovered
and
analyzed
by
Western
blotting.

Input
refers
to
3.3%
of
the
initial
reaction
volume.

(F)
Egg
extract
was
either
mock

depleted
(“mock”),
or
immunodepleted
of
TopBP1
using
TopBP1
antibodies

(TopBP1
-‐
).

TopBP1-‐depleted
extract
was
then
supplemented
with
either
blank
IVT

(-‐),
or
IVTs
expressing
the
indicated
myc-‐tagged
TopBP1
protein.

The
samples
were

then
blotted
with
antibodies
against
TopBP1.
(G)
The
egg
extracts
from
(A)
were

supplemented
with
sperm
chromatin
and,
optionally,
EcoRI
to
produce
DSBs
(“+

DSB”).

After
incubation,
the
sperm
chromatin
was
isolated
as
described
(Yan
et
al,

2009)
and
the
samples
blotted
with
either
Mab
9E10,
to
detect
myc-‐tagged
TopBP1,

or
antibodies
against
Orc2,
as
a
loading
control
(panel
“chromatin
fraction”).

In

addition,
samples
of
the
total
extract
were
blotted
for
S345-‐phosphorylated
Chk1

(Chk1-‐P)
or
Chk1
as
a
loading
control
(panel
“total
extract”).

39

Figure 3!
Rad9!
binding!
0! 1! 2!
RPA-ssDNA!
binding!
E!
GST-Rad9-T+!
TopBP1 BRCT:!
1! 2! 0-2!
BOUND!
INPUT!
D!
BRCT2 + RPA-ssDNA!
75! 100! 150! 50! (nt)!
INPUT!
BOUND!
C!
WT! W265R!
- +! - +!
GST-BRCT2 + ssDNA!
RPA:!
INPUT!
BOUND!
TopBP1 + ssDNA!
BOUND!
INPUT!
- +! RPA:!
WT!
- +!
W265R!
B!
WT!
W265R!
A!
myc-TopBP1 + XEE +:!
Chk1-P!
TopBP1
-
extract +:!
WT! W265R! -!
mock!
DSB:! -! +! +! +! +!
Chk1!
G!
myc-!
TopBP1!
Orc2!
chromatin fraction!
total extract!
F!
TopBP1
-
extract +:!
TopBP1!
WT! W265R! -! mock!

40

Figure
6.

ATR
activity
limits
TopBP1
chromatin
recruitment.
(A)
Egg
extracts

were
supplemented
with
sperm
chromatin
and
optionally
EcoRI
to
produce
DSB’s,

ATM,
ATR,
or
ATM/ATR
inhibitors.
After
one
hour
incubation,
the
sperm
chromatin

was
isolated
by
layering
it
on
top
of
30%
sucrose
+
NIB
(refer
to
Materials
and

Methods
for
recipe)
solution,
spun
down,
and
washed
three
times
with
NIB.

Samples
were
processed
for
Western
blot
and
then
probed
with
antibodies
against

endogenous
TopBP1
or
orc2
(as
loading
control).
(B)
Xenopus
egg
extracts
were

incubated
optionally
with
linearized
plasmid
DNA
to
introduce
DSB’s
and
optionally

ATM,
ATR,
or
ATM/ATR
inhibitors.
After
a
30
minute
incubation,
samples
were

resolved
on
an
SDS
gel
where
the
conditions
were
optimized
to
detect
a

phosphorylation
shift
and
prepared
for
Western
blot.
Blots
were
probed
with

antibodies
against
endogenous
TopBP1
and
Mab
9E10,
which
recognizes
the
myc

tag.
Inhibition
of
ATR
activity
by
ATRi
promotes
TopBP1
chromatin
association

under
normal
and
damage
conditions.

These
data
suggest
the
possibility
of
a

negative
feedback
loop,
whereby
once
ATR
is
activated
it
can
suppress
the
further

recruitment
of
TopBP1.

Figure 4 !
Orc2!
chromatin fraction!
TopBP1!
+DSB!
+DSB!
+ATMi!
+DSB!
+ATRi! +ATRi!
XEE + sperm chromatin +:!
A!
ATR!
ATRIP!
TopBP1!
Rad17!
B!
5’!
TopBP1!
TopBP1!
Rad17!
9-1-1!
ATR!
ATRIP!
Rad17!
ATR!
ATRIP!
TopBP1!
TopBP1!
5’!
TopBP1!
Rad17!
ATR!
ATRIP!
TopBP1!
TopBP1 senses stalled fork!
TopBP1 recruits Rad17!
9-1-1 loading stabilizes TopBP1!
ATR activated!
1! 2! 3! 4! 5!
!"#$%&'
'
'
())''''''''''''''*'''''''''''''''+''''''''''''''''+'''''''''''''+''''''''''''''''+''''''''''',-.'
/!01''''''/!21''''''''.3!4'
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!"#$%!!!!!"#&'!
())!!!!!!!!!!#*+,!!!!!!!!!!-./-0!!!!-./-0!!!!-./-0!!!!!!!-./-0!!!!!-.1-/!!!!!-.-20!!!!!-.-/0!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
3)!4*56$+!789!:;<=4!789!65>?@!
A=;B'1!
.'
/'
!"#$$
$
$
%&'#()$
B

A

41

Figure
7.

Revised
model
for
ATR
activation
during
checkpoint
response.

Please
refer
to
text
for
details.

Figure 4 !
Orc2!
chromatin fraction!
TopBP1!
+DSB!
+DSB!
+ATMi!
+DSB!
+ATRi! +ATRi!
XEE + sperm chromatin +:!
A!
ATR!
ATRIP!
TopBP1!
Rad17!
B!
5’!
TopBP1!
TopBP1!
Rad17!
9-1-1!
ATR!
ATRIP!
Rad17!
ATR!
ATRIP!
TopBP1!
TopBP1!
5’!
TopBP1!
Rad17!
ATR!
ATRIP!
TopBP1!
TopBP1 senses stalled fork!
TopBP1 recruits Rad17!
9-1-1 loading stabilizes TopBP1!
ATR activated!
1! 2! 3! 4! 5!

42

Chapter
4

Analysis
of
TopBP1
structure
regulation
during
checkpoint
signaling

43

Abstract

TopBP1
has
many
functions
inside
of
cells
so
which
protein
partner
it
binds

to
and
when
must
be
under
strict
control
by
the
cell
cycle.
One
way
to
do
this
that

does
not
require
the
limiting
of
its
binding
partners
during
the
cell
cycle
is
control
of

its
overall
structure,
since
how
this
is
controlled
can
become
important
in

determining
which
partners
it
will
favor
over
others.

It
is
known
that
TopBP1

structure
is
controlled
directly
by
AKT
through
phosphorylation,
so
in
cancers
with

high
AKT
activity,
AKT-‐mediated
TopBP1
dimerization
enhances
its
role
in

preventing
apoptosis
while
also
preventing
its
role
in
checkpoint
signaling.

TopBP1

function
is
also
controlled
by
the
availability
of
nutrients,
since
limited
nutrients

leads
to
its
de-‐acetylation,
which
prevents
its
role
in
DNA
replication
and
checkpoint

signaling.

Here
we’ve
identified
and
studied
a
point
mutant,
BRCT4/5
WR,
that
seems

to
have
a
different
structure
to
the
wild-‐type
protein
and
strongly
enhances
the

checkpoint
in
the
absence
of
DNA
damage
or
even
DNA.

This
is
possibly
due
to
a

greater
accessibility
of
its
AAD
to
ATR,
but
further
work
is
needed
to
confirm
this
or

any
other
possibility.

Given
the
fact
that
this
mutant
strongly
enhances
the

checkpoint,
it
might
in
the
future
prove
useful
in
allowing
us
to
gain
a
clearer

understanding
of
the
changes
TopBP1
normally
undergoes
in
the
process
of

checkpoint
activation
and
might
be
important
in
further
describing
the
order
in

sequence
of
events
that
lead
to
checkpoint
activation.

44

4.1
Introduction

TopBP1
is
required
for
many
processes
inside
a
cell.
Thus,
how
its
structure
is

regulated
to
affect
its
function
in
its
many
roles
has
been
studied,
since
its
proper

control
may
be
essential
to
prevent
breast
cancer
development
(Pedram,
2009;

Chowdhury,
2014).
TopBP1
controls
E2F1
and
p53
activities
through
a
direct

interaction
(Liu,
2009;
Liu,
2003).
Studies
show
that
77%
of
all
breast
cancers
have

a
genetic
alteration
in
PI3K/AKT
pathway,
while
nearly
half
have
altered
p53

(Chowdhury,
2014).

This
is
important
because
a
significant
portion
of
solid
tumors

show
deregulation
in
these
common
pathways.
For
example,
many
of
these
changes

in
tumors
result
in
hyper-‐activation
of
AKT
activity,
which
prevents
apoptosis

(Chowdhury,
2014;
Xu,
2012).
AKT
phosphorylation
of
S1159
on
human
TopBP1

causes
TopBP1
oligomerization,
mediated
by
BRCT
7-‐8
on
TopBP1
(Liu,
2013;
Liu,

2006).
TopBP1
oligomers
are
able
to
bind
E2F1
to
inactivate
its
pro-‐apoptotic

ability
to
allow
cancerous
cells
to
survive
(Liu,
2006).

In
addition,
this

phosphorylation
prevents
TopBP1
chromatin
association
and
thus
inhibits

checkpoint
activation,
promoting
genomic
instability
(Chowdhury,
2014).

After

many
years
of
research
by
this
group
into
understanding
how
AKT
affects
TopBP1

function
by
regulating
its
structure,
a
recent
study
shows
how
directly
targeting

TopBP1
and
modulating
its
activity
by
novel
compounds,
calcein
and
others
which

they
hope
to
develop,
can
potentially
lead
to
new
advances
in
cancer
therapy

(Chowdhury,
2014).
Thus,
understanding
how
this
protein
is
controlled
and

regulated
by
changes
to
its
overall
structure
is
important.

Other
work
in
human
cell
lines,
has
also
recently
explored
how
TopBP1

45

structure
is
controlled
under
different
metabolic
conditions
to
affect
its
function.

The
SIRT1
deacetylase
is
an
important
regulator
of
energy
homeostasis,
where
its

activity
is
promoted
by
glucose
starvation
or
caloric
restriction
but
negatively

controlled
by
DNA
damage
and
oxidative
stress
(Liu,
2014).
A
screen
identified
that

the
acetyltranferases
P300/CBP
acetylate
TopBP1
at
the
same
residues
where

SIRT1
deacetylates
TopBP1.
They
suggest
a
model
where
the
active
monomeric

form
of
TopBP1
allows
DNA
replication
and
enhances
the
checkpoint;
this,
they

conclude,
is
accomplished
by
acetylation
of
TopBP1
at
several
residues,
the
main

ones
being
K475,
789,
825.
They
found
that
active,
acetylated
TopBP1
monomer

lead
to
increased
binding
to
Treslin,
resulting
in
increased
cdc45
loading
and

subsequent
DNA
replication.
Checkpoint
response
to
HU
is
also
positively
affected

by
acetylation
in
increasing
TopBP1/Rad9
association
and
ultimately
Chk1

activation.
Deacetylation
led
to
TopBP1
oligomerization
and
prevented
its
function

in
both
replication
and
checkpoint.

TopBP1’s
role
in
checkpoint
activation
is
as
a
scaffold
and
activator
protein
that

recruits
checkpoint
components
to
DNA,
and
together
with
these,
activates
ATR

kinase
ability.
TopBP1
contains
an
ATR
Activating
Domain
(AAD)
that
when
directly

contacting
ATR,
activates
its
kinase
ability
(Kumagai,
2006).
Although
the
full-‐length

TopBP1
protein
is
a
poor
ATR
activator
in
the
absence
of
DNA,
a
small
fragment
of

the
protein
containing
the
AAD
of
TopBP1
is
a
potent
activator
of
ATR
in
solution

even
in
the
absence
of
DNA
(Kumagai,
2006).
However,
the
exact
mechanism
by

which
TopBP1’s
AAD
is
revealed
to
make
contact
with
ATR
on
DNA
is
not
fully

understood.

Data
has
shown
that
TopBP1’s
ability
to
bind
to
Rad9
is
important
for

46

its
ability
to
make
contact
with
ATR-‐ATRIP
(Lee,
2007).

Experiments
show
that

BRCT
1-‐2
of
TopBP1
mediate
binding
between
Rad9
and
TopBP1
and
that
a
deletion

mutant
in
these
domains
prevents
TopBP1-‐911
complex
association
to
ATR/ATRIP

on
the
DNA,
which
ultimately
prevents
checkpoint
activation
(Lee,
2007).

They

speculate
that
binding
of
TopBP1
to
the
911
complex
on
DNA
triggers
a

conformational
change
in
TopBP1
that
allows
exposure
of
its
AAD
to
ATR-‐ATRIP.

Here
we
identify
a
TopBP1
mutant
that
is
able
to
cause
an
increase
in
ATR

activation,
even
in
the
absence
of
any
DNA,
and
explore
the
possibility
that
this

phenotype
is
due
to
changes
in
its
structure
that
allow
the
AAD
to
be
exposed

without
the
need
for
TopBP1
to
be
on
DNA.
Hence,
this
mutant
seems
to
be
stuck
in

a
conformation
that
mimics
the
conformation
TopBP1
normally
undergoes
when
it

is
serving
in
its
role
in
activating
the
checkpoint.

47

4.2
Results

A
TopBP1
4/5WR
mutant
leads
to
increased
checkpoint
signaling

We
were
interested
in
the
role
the
BRCT4-‐5
region
of
TopBP1
plays
in
ATR

activation.

To
study
this,
we
decided
to
introduce
point
mutations
in
a
conserved

tryptophan
residue
in
each
BRCT
domain
(W603
in
BRCT4
and
W708
in
BRCT5)
to

change
them
into
arginine
to
destabilize
these
domains.
For
the
rest
of
the
study,

this
mutant
will
be
referred
to
as
BRCT4/5
WR
mutant.
We
then
decided
to
test
the

functional
significance
of
these
mutations
in
checkpoint
signaling
using
egg
extracts.

To
do
this,
Xenopus
egg
extracts
were
supplemented
with
sperm
chromatin,
myc-‐
tagged
full-‐length
either
wild
type
or
with
BRCT
4/5
WR
mutant
IVT
TopBP1

protein
and
optionally
aphidicolin
(10uM)
to
inhibit
polymerase
activity.
Reactions

were
incubated
for
30
minutes
at
room
temperature
and
a
sample
of
the
whole

reaction
was
taken
and
processed
for
Western
blot.
Surprisingly,
we
see
an
over

fourfold
increase
in
ATR
activation,
as
measured
by
increased
Chk1

phosphorylation,
when
the
BRCT
4/5
WR
mutant
is
added,
with
a
slight
increase

observed
even
in
conditions
where
no
DNA
damage
is
present
versus
wild
type

(Figure
8A).

To
test
to
see
if
these
mutations
have
any
effect
on
DNA
replication
as

an
increase
in
origin
firing
can
also
lead
to
an
increase
in
ATR
activation,
we
used

egg
extracts
supplemented
with
sperm
chromatin,
myc-‐tagged
TopBP1
full-‐length

wild
type
or
with
BRCT
4/5
WR
mutant
and
radiolabeled
dATP
P
32

to
allow

detection
of
newly
synthesized
DNA
and
looked
for
any
changes
in
signal
during
the

first
30
minutes.
We
find
that
BRCT4/5
WR
does
not
affect
replication
when
added

48

under
the
same
conditions
as
Figure
8A
above,
showing
addition
of
TopBP1

BRCT4/5
WR
mutant
does
not
lead
to
increased
replication
at
early
origins
(Figure

8B).

Thus,
an
increase
in
DNA
replication
does
not
explain
the
strong
increase
in

checkpoint
activation
phenotype
we
observed.

Because
we
saw
an
increase
in
ATR
activation
when
we
added
TopBP1

BRCT4/5
WR
mutant
to
extract,
we
wanted
to
find
an
explanation
for
this
ability.

First,
we
reasoned
that
this
might
be
due
to
an
enhanced
ability
to
bind
sites
of

replication
stress.
To
test
this
possibility,
Xenopus
egg
extracts
were
supplemented

with
sperm
chromatin,
myc-‐tagged
full-‐length
wild
type
or
BRCT
4/5
WR
IVT

TopBP1
protein,
and
optionally
aphidicolin
(10uM
or
300uM)
as
indicated
to
inhibit

polymerase
activity.
Reactions
were
incubated
again
for
30
minutes,
same
as

conditions
above,
at
room
temperature.
After
incubation,
the
sperm
chromatin
was

isolated
and
chromatin
bound
proteins
analyzed
by
Western
blot.
We
see
that
the

BRCT4/5
WR
mutant
does
not
bind
better
to
stalled
forks
compared
to
wild
type

(Figure
9A).
We
therefore
conclude
that
binding
better
to
stalled
forks
is
not
the

reason
4/5
WR
mutant
hyper-‐activates
ATR.

To
try
to
determine
if
the
increase
in

ATR
signaling
was
due
to
enhanced
checkpoint
complex
assembly,
we
looked
at

whether
addition
of
excess
BRCT
4/5
WR
mutant
TopBP1
was
able
to
increase

recruitment
of
checkpoint
complex
components
to
chromatin,
since
TopBP1
is

required
for
the
recruitment
of
Rad17
and
Rad9
to
chromatin
when
damage
is

present.
We
see
that
when
BRCT
4/5
WR
mutant
TopBP1
is
added,
no
increase
in

checkpoint
complex
components,
either
Rad
17
or
Rad9,
in
either
low
or
high

damage
conditions
is
seen
(Figure
9A).
To
confirm
this
result,
we
looked
at
the

49

ability
of
the
4/5WR
mutant
to
bind
the
phosphorylated
Rad9
tail.
To
do
this,
we

used
in
vitro
CK2-‐phosphorylated
Rad9
tail,
previously
purified
from
E.
coli,
and

incubated
this
with
either
wild
type
or
4/5
WR
TopBP1
IVT
proteins
at
30C
for
one

hour,
after
this
incubation
the
above
reactions
were
allowed
to
bind
to
glutathione

sepharose
beads
at
4C
for
one
hour,
samples
washed,
and
proteins
detected
by

Western
blot.
We
see
that
BRCT
4/5
WR
mutant
TopBP1
binds
less
well
than
WT
to

purified
phosphorylated
Rad9
tail
(Figure
9B;
Experiment
performed
by
Matt

Michael).

Thus,
the
mechanism
of
enhanced
increased
checkpoint
signaling
is
likely

not
due
to
increase
checkpoint
complex
assembly.

BRCT4/5
WR
does
not
hyper-‐accumulate
on
chromatin
in
the
absence
of
ATR

activity
and
can
activate
ATR
in
the
absence
of
DNA
damage

As
shown
in
Chapter
3,
Figure
8B,
ATR
activity
limits
TopBP1
chromatin

association
so
we
therefore
wanted
to
know
the
BRCT4/5
WR
mutant
behaved
in
a

similar
way.
Sperm
chromatin
was
added
to
egg
extract,
supplemented
with
either

wild-‐type
or
4/5WR
mutant
TopBP1
and
the
samples
were
optionally
treated
with

chemical
inhibitors
of
either
ATM
or
ATR
(ATMi
and
ATRi,
respectively)
and
EcoRI

to
produce
DSBs.

After
incubation
the
chromatin
was
isolated
and
probed
for

TopBP1
and
Orc2,
as
a
loading
control.

WT
TopBP1
shows
an
increase
in
chromatin

binding
in
response
to
double
strand
breaks,
and
this
chromatin
accumulation
is

further
enhanced
in
the
presence
of
an
ATR
inhibitor
(Figure
10).

In
contrast,
we

see
that
BRCT
4/5
WR
mutant
TopBP1
does
not
hyper-‐accumulate
in
the
presence

of
an
ATR
inhibitor
and
again
see
more
clearly
that
no
DNA
damage
is
required
to

50

see
an
enhanced
ATR
activation,
as
seen
by
phosphorylated
Chk1
signaling.

This

suggests,
that
although
ATR
activity
normally
inhibits
the
amount
of
wild
type

TopBP1
on
chromatin,
the
BRCT4/5WR
mutant
does
not
respond
to
this
inhibition

and
seems
to
act
in
a
manner
that
does
not
require
its
presence
on
chromatin
to

activate
the
checkpoint.

The
4/5
WR
mutant
is
able
activate
ATR
in
the
absence
of
DNA

Since
we
see
that
BRCT
4/5
WR
mutant
does
not
show
enhanced
chromatin

association
even
when
ATR
activity
is
inhibited,
checkpoint
complex
assembly,
or

Rad9
binding,
we
wondered
if
DNA
was
even
necessary
for
the
ability
of
the
4/5
WR

mutant
to
hyperactivate
ATR.
For
this,
Xenopus
egg
extracts
were
incubated

optionally
with
linearized
plasmid
DNA
to
introduce
DSB’s
as
a
positive
control,
or

simply
supplemented
with
myc-‐tagged
full-‐length
wild
type
or
4/5WR
mutant
IVT

TopBP1
proteins
with
no
added
DNA
at
all.
After
room
temperature
incubation
at

indicated
time
points,
samples
were
taken
and
processed
for
Western
blots.
We
see

TopBP1
BRCT4/5
WR
mutant
is
in
fact
able
to
activate
ATR
independently
of
any

DNA,
but
only
after
around
60
minutes,
where
previously
we
saw
a
strong
effect
as

early
as
30
minutes
when
DNA
was
present
(Figure
11).

We
therefore
decided
to
see
if
by
inhibiting
de-‐phosphorylation
we
would
be

better
able
to
study
the
phenotype
of
the
TOPBP1
mutant
without
DNA
we

observed.
We
used
Xenopus
egg
extracts
incubated
optionally
with
linearized

plasmid
DNA
to
introduce
DSB’s,
an
ATR
inhibitor,
and/or
okadaic
acid
(OA)
at
a

51

concentration
that
preferentially
inhibits
the
PP2A
phosphatase,
as
indicated.
After

a
30
minute
incubation,
samples
were
processed
for
Western
blot.
We
see
that
in

the
presence
of
OA,
the
checkpoint
response
is
greatly
increased
after
linearized

plasmid
is
added,
thus
this
shows
that
this
method
can
allow
us
to
look
at
the
slight

changes
in
checkpoint
activation
the
mutant
exhibits
in
a
more
consistent
manner

(Figure
12A).

We
then
next
tested
wild
type
and
BRCT
4/5
WR
mutant
IVT
proteins

under
the
same
conditions
and
experiment
set
up
as
above
and
looked
for
any

changes.
Like
we
saw
previously,
OA
leads
to
a
similar
increase
in
phosphorylated

Chk1
in
the
wild
type
and
mutant
after
damage,
but
the
mutant
showed
nearly
a

60%
increase
in
signal
in
the
absence
of
any
DNA
at
30
minutes,
a
lot
sooner
than

without
OA
we
saw
previously
after
an
hour
(Figure
12B).
First,
this
suggests
that

although
DNA
helps
amplify
the
signal
by
providing
a
scaffold
for
full
checkpoint

activation,
it
is
not
absolutely
necessary
for
checkpoint
activation
in
this
case,
as
the

mutant
does
not
require
DNA
for
its
effect
in
ATR
activation.
This
result
is

significant
since
it
shows
that
the
BRCT4/5WR
unlike
wild
type
is
able
to
directly

activate
the
checkpoint
in
the
absence
of
any
DNA.
Second,
this
result
suggests
that

the
effect
of
the
BRCT4/5WR
mutant
in
ATR
activation
likely
has
something
to
do

with
a
conformational
change
when
compared
to
wild
type.
Evidence
that
the
4/5

mutant
is
conformationally
different
than
the
wild-‐type
can
be
gained
from
the
fact

the
mutant
protein
does
not
bind
Rad
9
as
well
as
the
wild
type.

We
suspect
that
the

BRCT4/5
WR
mutant
is
locked
in
a
conformation
that
the
wild
type
protein
only

assumes
when
it
is
on
the
DNA
to
allow
it
to
activate
ATR.

To
gain
further
evidence
that
the
BRCT4/5
WR
mutant
is
conformationally

52

different
than
the
wild-‐
type
protein,
we
asked
whether
the
BRCT
4/5
WR
mutant

shows
a
different
phosphorylation
pattern
than
wild-‐type
in
response
to
ATR

activity.

To
test
this,
Xenopus
egg
extracts
were
supplemented
with
TopBP1
either

wild-‐type
or
indicated
mutant
and
incubated
with
or
without
linearized
plasmid

DNA
to
introduce
a
DSB
for
checkpoint
activation.
After
a
30
minute
incubation,

samples
were
resolved
on
a
SDS
gel
where
the
conditions
were
optimized
to
detect
a

phosphorylation
shift
and
prepared
for
Western
blot.
Wild
type
TopBP1
shows

damage
dependent
shift,
in
contrast,
BRCT2
265WR
mutant
does
not
get

phosphorylated
after
damage.
This
could
be
due
to
its
inability
to
bind
to
chromatin

and
get
phosphorylated
there
or
because
its
in
a
state
that
covers
up
the
sites
that

get
phosphorylated
after
damage.
TopBP1
BRCT4/5WR
lacks
some
damage

independent
phosphorylation,
but
still
retains
some
damage
dependent

phosphorylation,
as
seen
by
the
fact
that
it
migrates
faster
than
wild-‐type
under
no

damage
conditions
although
nothing
close
to
the
wild
type
(Figure
13).
This

suggests
that
the
mutations
on
BRCT4/5
WR
make
it
unable
to
get
easily
modified

after
damage,
implying
a
conformational
change
where
the
ATR
phosphor-‐acceptor

sites
are
hidden.

These
data
suggest
that
the
4/5
WR
mutant
assumes
a
very
different

conformation
than
the
wild-‐type
protein.

How
might
this
be
happening?
As

mentioned
earlier,
previous
work
has
shown
that
TopBP1
can
self-‐associate
and

that
self-‐association
regulates
its
function.
We
wondered
whether
perhaps
the

conformation
difference
between
wild-‐type
and
4/5
WR
is
due
to
changes
in
self-‐
association.
Thus,
we
decided
to
test
whether
there
is
any
difference
in
self-‐

53

association
between
wild-‐type
and
4/5
WR
mutant.

To
do
this,
myc-‐tagged
and
his-‐
tagged
full
length
TopBP1
either
wild
type
or
BRCT4/5
WR
mutant
were
produced

by
IVT
and
then
mixed
and
incubated
for
one
hour
at
room
temperature
to
allow

oligomerization.
Beads
coupled
with
and
without
his-‐antibodies
were
prepared
and

then
these
were
combined
with
binding
buffer
and
reactions
above
for
2hrs
at
4C

with
rotation
for
immunoprecipitation,
then
after
incubation
washed
and
samples

processed
for
Western
blot.
A
preliminary
pull-‐down
experiment
of
his
tagged

proteins
(either
wildtype
or
BRCT
4/5
WR
mutant
as
described)
show
that
although

wild
type
TopBP1
is
capable
of
forming
oligomers
with
itself,
BRCT4/5
WR

mutations
lead
to
an
enhancement
in
this
oligomerization
ability
(Figure
14).

Thus,

it
seems
possible
that
the
phenotype
of
the
mutant
can
be
explained
by
its
ability
to

better
oligomerize
such
that
two
proteins
binding
together
can
reveal
the
AAD.

54

4.3
Discussion

Although
we
still
don’t
completely
understand
the
full
details
of
how
TopBP1

is
controlled
during
a
checkpoint
response
and
what
conformational
changes
it

normally
undergoes
during
and
after
ATR
activation,
the
study
of
the
4/5WR

mutant
seems
promising
in
allowing
us
to
gain
a
clearer
understanding
of
this
event.

Our
initial
observation
that
the
BRCT
4/5
WR
mutant
led
to
a
significant
increase
in

ATR
activation
after
damage
in
response
to
stalled
forks
led
us
to
want
to
study
it

further.

We
saw
that
when
we
add
BRCT
4/5
WR
mutant
to
extract,
it
seems
that

neither
a
great
increase
in
DNA
replication
nor
an
increase
in
checkpoint
complex

assembly
were
able
to
explain
the
phenotype.
In
fact,
we
see
that
the
mutant
seems

to
bind
less
readily
to
the
phosphorylated
Rad9
tail,
which
is
the
main
binding
site

between
TopBP1-‐911
for
checkpoint
complex
assembly,
in
pull
down
experiments.

Typically,
when
we
add
an
ATR
inhibitor
to
extract,
we
see
a
big
increase
in

TopBP1
chromatin
association
under
normal
conditions,
double-‐strand
breaks,
or

stalled
forks,
suggesting
that
ATR
activity
normally
controls
the
amount
of
TopBP1

on
chromatin,
yet
we
lack
to
see
this
hyper-‐accumulation
with
the
BRCT4/5
WR

mutant.
Importantly,
we
see
that
the
mutant
is
able
to
activate
ATR
even
in
the

absence
of
any
DNA.
Whereas
we
see
ATR
activation
by
the
mutant
after
around
an

hour
without
any
DNA
present,
when
we
add
OA
to
inhibit
phosphatases,
we
see
an

earlier
activation
at
30
minutes.
Additionally,
when
we
look
at
the
phosphorylation

status
of
the
mutant
in
comparison
to
wild
type,
we
see
there
is
a
loss
in
post-‐
translational
modification
after
damage.
Altogether,
these
data
suggest
that
the
4/5

WR
mutant
is
in
a
conformation
or
state
that
mimics
the
physiological
state
TopBP1

55

is
in
when
ATR
activity
is
high.

The
available
data
thus
suggest
a
couple
of
possibilities
for
how
the
mutant

can
lead
to
an
increase
in
ATR
activation:
(1)
Model
1-‐
transition
from
monomer
to

oligomer
and
(2)
Model
2-‐
lack
of
negative
feedback
by
ATR.

For
Model
1
(monomer

versus
oligomer)
to
be
correct,
we
would
expect
the
wild-‐type
TopBP1
to
be

monomeric
in
solution.

The
monomeric
form
would
have
a
hidden
AAD
and

exposed
ATR
phosphor-‐acceptor
sites.

Perhaps
the
AAD,
in
this
case,
would
be

hidden
through
intra-‐molecular
interactions.
During
a
checkpoint
response,
the
wild

type
protein
transitions
from
monomer
to
oligomer
only
when
it
is
bound
to
DNA.

By
contrast,
in
solution
4/5
WR
is
oligomeric.

The
oligomeric
form
has
an
exposed

AAD
and
hidden
ATR
phosphor-‐acceptor
sites.

To
accept
this
model,
it
will
be

necessary
to
confirm,
with
better
experiments,
that
4/5
WR
does
indeed
self-‐
associate
more
readily
than
WT.

If
this
is
the
case,
future
experiments
can
be
done

to
determine
the
domains
within
the
4/5
WR
mutant
that
are
required
for
self-‐
association.
Then,
it
will
be
possible
to
identify
mutations
that
prevent
the
4/5
WR

mutant
from
self-‐associating
and
ask
if
these
mutants
can
still
activate
ATR
in
a

DNA-‐independent
manner.
These
mutations
could
then
be
made
in
the
wild
type

and
used
to
ask
if
it
too
can
still
activate
ATR
during
a
checkpoint
response,
to
show

that
dimerization
is
essential
for
ATR
activation
by
TopBP1.

For
model
2
(negative
feedback
by
ATR),
it
can
be
possible
that
the
wild
type

protein
can
indeed
activate
ATR
in
solution,
but
it
is
then
immediately

phosphorylated
by
ATR
to
prevent
further
activation
through
a
negative
feedback

loop.
If
this
is
the
case,
the
4/5
WR
mutant
cannot
be
phosphorylated
by
ATR,
and

56

thus
there
is
no
negative
feedback,
and
ATR
gets
hyper-‐activated.
To
test
this
model,

it
would
be
necessary
to
identify
the
ATR
phospho-‐acceptor
sites
in
TopBP1
and

mutate
them.
Then
the
question
can
be
asked
if
the
ATR
phosphor-‐acceptor
mutant

can
activate
ATR
in
a
DNA-‐independent
manner.
Irrespective
of
which
of
these
two

models
proves
to
be
correct,
the
study
of
this
mutant
will
allow
a
better

understanding
of
how
TopBP1
is
controlled
during
its
role
in
ATR
activation.

57

Figure
8.
TopBP1
4/5WR
mutant
leads
to
an
enhanced
checkpoint
response

that
cannot
be
explained
by
an
increase
in
DNA
replication.
(A)
Xenopus
egg

extracts
were
supplemented
with
sperm
chromatin,
myc-‐tagged
full-‐length
either

wild
type
or
with
the
4/5
WR
mutant
IVT
TopBP1
protein
or
IVT
“blank”
lysate

(negative
control),
and
optionally
aphidicolin
(10uM)
to
inhibit
polymerase
activity.

Reactions
were
incubated
for
30
minutes
at
room
temperature
and
sample
of
whole

reaction
was
taken
and
processed
for
Western
blots.

Checkpoint
activation
was

measured
by
looking
at
amount
of
phosphorylated
Chk1
using
antibodies
specific

for
serine
345-‐
phosphorylated
Chk1
(Chk1-‐P)
and
Chk1
(as
loading
control).

TopBP1
was
detected
using
Mab
9E10,
which
recognizes
the
myc
tag.
(B)
Xenopus

egg
extracts
were
supplemented
with
sperm
chromatin,
myc-‐tagged
TopBP1
full-‐
length
wild
type
or
with
the
4/5
WR
mutant
and
radiolabeled
dATP
p32
to
allow

detection
of
newly
synthesized
DNA.
After
indicated
times,
samples
were
collected,

treated
with
proteinase
K,
to
remove
chromatin
associated
proteins,
for
1
hour
at

37C
and
then
resolved
on
an
agarose
gel.

Gel
was
then
dried,
exposed
to
a

phosphoscreen,
and
developed.

Chk1-P!
Chk1!
blank!
WT!
TopBP1!
Aphidicolin!
myc-TopBP1!
- +! - +! - +! -!
4/5 WR!
TopBP1!
A!
Figure 4!
E!
10! 15! 20! 25! 30! 35! 10! 15! 20! 25! 30! 35! (min.)!
WT! W603R,W708R!
32
P!
C!
GST-Rad9-T+!
TopBP1!
BOUND!
INPUT!
WT!
W603R!
W708R!
A!
FL TopBP1 + ssDNA!
BOUND!
INPUT!
- +! RPA:!
WT!
- +! - +!
W603R!
W708R!
W265R!
W603R!
W708R!
D!
Chk1-P!
Chk1!
WT!
Aphidicolin!
myc-TopBP1!
- +! - +!
W603R!
W708R!
relative amount!
of Chk1-P signal!
1! 13! 3! 53!
TopBP1!
B!
myc-TopBP1!
RPA (32)!
Orc2!
isolated chromatin!
WT!
W603R!
W708R!
TopBP1
-
XEE + aphid.!
W265R!
myc-TopBP1!
total extract!
Chk1-P!
Chk1!
B!
WT TopBP1! 4/5 WR TopBP1!

58

Figure
9.
TopBP1
BRCT4/5
WR
mutant
does
not
enhance
checkpoint
complex

assembly.
(A)
Xenopus
egg
extracts
were
supplemented
with
sperm
chromatin,

myc-‐tagged
full-‐length
wild
type
or
with
BRCT4/5
WR
IVT
TopBP1
protein,
and

optionally
aphidicolin
(10uM
or
300uM)
as
indicated
to
inhibit
polymerase
activity.

Reactions
were
incubated
for
30
minutes
at
room
temperature.
After
incubation,
the

sperm
chromatin
was
isolated
by
layering
it
on
top
of
30%
sucrose
+
NIB
(refer
to

Materials
and
Methods
for
recipe)
solution,
spun
down,
and
washed
three
times

with
NIB
and
analyzed
by
Western
blot.

Chromatin
bound
proteins
were
then

probed
with
specific
antibodies:
Mab
9E10,
which
recognizes
the
myc
tag,
Rad17,

Rad9,
RPA32,
Orc2
(as
loading
control).
(B)
Myc-‐tagged,
full-‐length
TopBP1
either

wild
type
or
indicated
point
mutations
were
produced
by
IVT
and
then
mixed
with

purified
GST-‐Rad9T
previously
phosphorylated
by
CK2
and
incubated
for
one
hour

at
30C.
Beads
coupled
with
GST-‐antibodies
were
prepared
and
then
these
were

combined
with
binding
buffer
and
reactions
above
for
one
hour
at
4C
with
rotation.

After
incubation,
beads
were
pelleted
by
centrifugation,
washed,
eluted,
and

proteins
detected
by
Western
blot.

The
TopBP1
fragments
were
detected
anti-‐GST

antibodies.

59

A!
!"#$%&$
'()*)%$
!"#$+$
(!,-$
!)"$.-$
$$/$$$$$$$$$%/$$$$$$$.//$$$$$$$$$$/$$$$$$$$$$$%/$$$$$$$.//$$$$$$01$")2$$
3'$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$14'"5'$
GST-Rad9-T+!
FL TopBP1!
BOUND!
INPUT!
WT!
!
4/5 WR!
B!
A!
!"#$%&$
'()*)%$
!"#$+$
(!,-$
!)"$.-$
$$/$$$$$$$$$%/$$$$$$$.//$$$$$$$$$$/$$$$$$$$$$$%/$$$$$$$.//$$$$$$01$")2$$
3'$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$14'"5'$
GST-Rad9-T+!
FL TopBP1!
BOUND!
INPUT!
WT!
!
4/5 WR!
B!

60

Figure
10.
TopBP1
BRCT4/5
WR
mutant
does
not
respond
to
ATR
activity
as

the
wild
type
and
shows
no
enhanced
recruitment
to
sites
of
damage.

Xenopus

egg
extracts
were
supplemented
with
sperm
chromatin,
IVT
myc-‐tagged
TopBP1
or

4/5WR
mutant,
and
incubated
optionally
with
EcoRI
to
introduce
DSB’s
and
an
ATR

inhibitor,
as
indicated.
After
a
30
minute
incubation,
samples
of
total
extract
were

taken
to
look
at
checkpoint
activation
and
the
sperm
chromatin
was
isolated
by

layering
it
on
top
of
30%
sucrose
+
NIB
(refer
to
Materials
and
Methods
for
recipe)

solution,
spun
down,
and
washed
three
times
with
NIB
and
samples
processed
and

analyzed
by
Western
blot.

Chromatin
bound
proteins
were
then
detected
with

specific
antibodies:
Mab
9E10,
which
recognizes
the
myc
tag
and
Orc2
(as
loading

control).
Checkpoint
activation
was
measured
by
looking
at
amount
of

phosphorylated
Chk1
in
total
extract
using
antibodies
specific
for
serine
345-‐

phosphorylated
Chk1
(Chk1-‐P)
and
Chk1
(as
loading
control).

Figure 5 !
DSB:!
ATRi:!
-!
-!
+!
-!
+!
+!
-!
-!
+!
-!
+!
+!
+OA!
Chk1-P!
(pSer345)!
D!
DSB:! - + - +! - + - +!
Chk1-P!
(pSer345)!
+OA! +OA!
WT!
W603R!
W708R!
TopBP1 + XEE +:!
E!
G!
Chk1-P!
(pSer345)!
mock XEE + OA:! TopBP1
-
XEE + OA:!
DSB:! - + - + - +!
WT!
W603R!
W708R!
W265R!
W603R!
W708R!
- + - + - +!
WT!
W603R!
W708R!
W265R!
W603R!
W708R!
F!
WT!
W603R!
W708R!
W265R!
W603R!
W708R!
-! mock!
TopBP1
-
XEE + IVT:!
TopBP1!
WT! W603,708R!
myc-TopBP1!
A!
ORC2!
myc-!
TopBP1!
chromatin-bound!
WT! W603,708R!
+DSB!
+ATRi!
+DSB! +DSB!
+ATRi!
+DSB!
total extract!
Chk1!
B!
Chk1-P!
(pSer345)!
myc-TopBP1 + XEE + SC:!
WT!
W603R!
W708R!
C!
WT!
W603R!
W708R!
Chk1-P!
(pSer345)!
Chk1!
buffer! + geminin!
myc-!
TopBP1!
DNA synthesis (a.u.)!
!"################################$%&#!'############################

61

.

Figure
11.

TopBP1
BRCT4/5
WR
mutant’s
ability
to
activate
the
checkpoint
is

not
dependent
on
damage
or
DNA.
Xenopus
egg
extracts
were
incubated

optionally
with
linearized
plasmid
DNA
to
introduce
DSB’s
as
a
positive
control,
or

simply
supplemented
with
myc-‐tagged
full-‐length
wild
type
or
4/5WR
mutant
IVT

TopBP1
protein
with
no
DNA.
After
room
temperature
incubation
at
indicated
time

points,
samples
were
taken
and
processed
for
Western
blots.
Checkpoint
activation

was
measured
by
looking
at
amount
of
phosphorylated
Chk1
in
total
extract
using

antibodies
specific
for
serine
345-‐
phosphorylated
Chk1
(Chk1-‐P)
and
Chk1
(as

loading
control).

!"########$%#########&%########!"#########$%########&%#######!"########$%########&%#########'()*+,-#
./01!#
#
#
12345!#
#####6#789#######################:;"<=######################<.#
!"#$%&'(
(
%)*+,-(
(
(
(
,.(/01-(

62

Figure
12.

TopBP1
BRCT4/5
WR
mutant
ability
to
activate
the
checkpoint
is

enhanced
by
the
addition
of
a
phosphatase
inhibitor.
(A)
Xenopus
egg
extracts

were
incubated
optionally
with
linearized
plasmid
DNA
to
introduce
DSB’s,

optionally
an
ATR
inhibitor,
and/or
okadaic
acid
(OA)
at
a
concentration
that

preferentially
inhibits
the
PP2A
phosphatase,
as
indicated.
After
a
30
minute

incubation,
samples
were
processed
for
Western
blot.
Checkpoint
activation
was

measured
by
looking
at
amount
of
phosphorylated
Chk1
in
total
extract
using

antibodies
specific
for
serine
345-‐
phosphorylated
Chk1
(Chk1-‐P).
(B)
Xenopus
egg

extracts
were
supplemented
with
myc-‐tagged
full-‐length
wild
type
or
4/5WR

mutant
IVT
TopBP1
and
incubated
optionally
with
linearized
plasmid
DNA
to

introduce
DSB’s
and
optionally
an
ATR
inhibitor
and
okadaic
acid
(OA)
at
a

concentration
that
preferentially
inhibits
the
PP2A
phosphatase,
as
indicated.
After

a
30
minute
incubation,
samples
were
processed
for
Western
blots.
Checkpoint

activation
was
measured
by
looking
at
amount
of
phosphorylated
Chk1
in
total

extract
using
antibodies
specific
for
serine
345-‐
phosphorylated
Chk1
(Chk1-‐P).

Percent
signal
for
Chk1-‐P
was
calculated
by
assigning
band
with
highest
intensity

100%
and
used
for
normalizing
rest
of
signals.

!"#$%&'
"''''''''('''''''''('''''''"'''''''''('''''''''''('
)*+,'
-./'01,2345'61478,9:'
(;)'
"''''''''"'''''''''(''''''''"'''''''''"''''''''''''('
B!
A!
"''''''''('''''''''"''''''''''''''(''''''''''''"''''''''''('''''''''''"''''''''''''(''''''''-./'
(;)' (;)'
'''''''<*''*;!/!&''''''''''''''''''''''''''''=>?<+'*;!/!&'
!"#$%&'
@'''''''''''ABC''''''''''''''''''''''''''''''D@C''''''&@@C'

63

Figure
13.
TopBP1
phosphorylation
status
in
response
to
damage
is
different

in
TopBP1
mutants.
Myc-‐tagged
wild
type,
W265R,
4/5WR
mutant
IVT
TopBP1

proteins
were
incubated
in
Xenopus
egg
extracts
for
30
minutes
in
the
presence
of

linear
DNA
to
introduce
DSB.
After
a
30
minute
incubation,
samples
were
resolved

on
an
SDS
gel
where
the
conditions
were
optimized
to
detect
a
phosphorylation
shift

and
prepared
for
Western
blot.
Blots
were
probed
with
antibodies
against
Mab

9E10,
which
recognizes
the
myc
tag.

Figure
14.
TopBP1
BRCT4/5
WR
shows
possible
enhanced
oligomerization.

Myc-‐tagged
and
his-‐tagged
full
length
TopBP1
either
wild
type
or
4/5
WR
mutant

were
produced
by
IVT
and
then
mixed
and
incubated
for
one
hour
at
room

temperature.
Beads
coupled
with
and
without
his-‐antibodies
were
prepared
and

then
these
were
combined
with
binding
buffer
and
reactions
above
for
1hr
at
room

temperature
with
rotation.
After
incubation,
beads
were
pelleted
by
centrifugation,

washed,
eluted,
and
proteins
detected
by
Western
blot.

The
TopBP1
fragments

were
detected
using
Mab
9E10,
which
recognizes
the
myc
tag.

Input
refers
to
1.6%

of
the
initial
binding
reaction.

!!"!!!!!!!!!!!#!!!!!!!!!!"!!!!!!!!!!!#!!!!!!!!!"!!!!!!!!!!!!!#!!!!!!!!!!"!!!!!!!!!!#!!!!!!!!!$%&!
!
!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!'()&*+!
!!!!!!,'!!!!!!!!!!!!!!!!!!,-./0!!!!!!!!!!!12/!,0!!!!!!!!!!!!!!!,'!!!!!!!!
3!
!
!
!
!
!
'()4*+!
!
!
566!!!!!!!!!!!"!!!!!!!!!!!!!!!#!!!!!!!!!!!!!!#!!!!!!!!!!!!!#!!!!!!!!!!!!!!!!#!!!!!!!!!!!$%&!
3'78!!!!!!!3'08!!!!!!!!&9':!
&!
!
+ ssDNA!
BOUND!
INPUT!
- +! RPA:!
WT!
- +! - +!
4/5 WR! 2/4/5 WR!
0! 1! 2! 3! 4! 5!
W603!W708! W265!
A!
BOUND!
INPUT!
WT!
+ RPA-ssDNA!
4/5 WR!
FL TopBP1!
B!
BOUND!
INPUT!
WT!
+ RPA-ssDNA!
!
4/5 WR!
BRCT4-5!
C!
!"#$%&'##
!"#()*#
+',#!-#./0120#$%&'##
+',#!-#./0120#()*#
##3##############4####################3############4#########$56#783891:6#
D!
!"#$%&'(&
!"""""""""#"""""""""!""""""""""#""""""""$%&""""""""""
'()*+"
"
"
,*%)-"
.-""""""""""""""/"012"
345"
"
345"
!"""""""""""""""""""""""""""""""""""""""""""""#"

64

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Asset Metadata

Creator Acevedo, Julyana (author)

Core Title Biochemical mechanism of TopBP1 recruitment to sites of DNA damage

Contributor Electronically uploaded by the author (provenance)

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Publication Date 11/16/2015

Defense Date 08/26/2015

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

Tag ATR activation,DNA damage,OAI-PMH Harvest,replication stress,TopBP1

Format application/pdf (imt)

Language English

Advisor Michael, Matthew (committee chair), Aparicio, Oscar (committee member), Chen, Lin (committee member), Curran, Sean (committee member), Forsburg, Susan (committee member)

Creator Email acejuli@gmail.com,julyanaa@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-199941

Unique identifier UC11277836

Identifier etd-AcevedoJul-4043.pdf (filename),usctheses-c40-199941 (legacy record id)

Legacy Identifier etd-AcevedoJul-4043.pdf

Dmrecord 199941

Document Type Dissertation

Format application/pdf (imt)

Rights Acevedo, Julyana

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Abstract (if available)

Abstract During a replication stress response or after double strand break resection, the ATR protein kinase is activated to allow a delay in cell cycle progression, a block to further origin firing, and stabilization of the stalled fork. ATR is recruited to stalled forks via an interaction between its binding partner ATRIP and RPA-coated single-stranded DNA (ssDNA). However, binding of ATR-ATRIP to RPA-coated ssDNA is not sufficient for ATR activation as both the 9-1-1 clamp protein and the checkpoint activator TopBP1 must also be recruited so that TopBP1 may activate ATR. The biochemical mechanism for assembly of the checkpoint-signaling complex has been an important unanswered question in the cell cycle field for many years. ❧ Previous work from our laboratory, and other groups, has shown that ATR/ATRIP and TopBP1/9-1-1 are recruited independently to stalled forks, and then come together on the DNA to form an active checkpoint-signaling complex. Furthermore, we have previously shown that TopBP1 is required for loading of the 9-1-1 clamp, however the means by which TopBP1 senses the stalled fork to allow checkpoint complex assembly was not previously known. Our data shows that direct interaction between TopBP1 BRCT repeat 2 and RPA-coated ssDNA is the mechanism for TopBP1 recruitment. Importantly, we determined that direct interaction with RPA-coated ssDNA is a general feature of a subclass of BRCT domains from other proteins, such as PTIP. Furthermore, we find that once TopBP1 has promoted initial ATR activation, further chromatin loading of TopBP1 to damaged sites is prevented by a negative feedback loop of ATR activity. ❧ Lastly, we’ve also identified and studied a point mutant, BRCT4/5 WR, that seems to have a different structure to the wild-type protein and strongly enhances the checkpoint in the absence of DNA damage or even DNA. This is possibly due to a greater accessibility of its AAD to ATR, but further work is needed to confirm this or any other possibility. Given the fact that this mutant strongly enhances the checkpoint, it might in the future prove useful in allowing us to gain a clearer understanding of the changes TopBP1 normally undergoes in the process of checkpoint activation and might be important in further describing the order in sequence of events that lead to checkpoint activation.