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STRUCTURAL STUDIES OF TWO KEY FACTORS FOR DNA REPLICATION IN
EUKARYOTIC CELLS
by
Yu-Hao Paul Chang
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
August 2010
Copyright 2010 Yu-Hao Paul Chang
Object Description
| Title | Structural studies of two key factors for DNA replication in eukaryotic cells |
| Author | Chang, Yu-Hao Paul |
| Author email | yuhaopch@usc.edu; yuhaopch@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Genetic, Molecular & Cellular Biology |
| School | Keck School of Medicine |
| Date defended/completed | 2010-04-20 |
| Date submitted | 2010 |
| Restricted until | Restricted until 06 Aug. 2012. |
| Date published | 2012-08-06 |
| Advisor (committee chair) | Chen, Xiaojiang |
| Advisor (committee member) |
Goodman, Myron Langen, Ralf Rees, Douglas |
| Abstract | Genomic DNA replication is essential for the transmission of genetic information; during this process, minichromosome maintenance (MCM) complex, the cellular replicative helicase, unwinds duplex DNA to enable DNA synthesis by polymerases. Eukaryotic DNA replication is a tightly regulated process and the recruitment of MCM to the replication origin and its activation require the participations of the GINS complex and more than ten additional DNA replication factors. My thesis project focuses on the structural studies of the GINS complex, as well as a MCM functional homolog, Simian virus 40 (SV40) large T antigen (LTag) helicase.; The crystal structure of the full-length human GINS hetero-tetramer was determined in order to further understand the functional role of GINS. The four subunits each has a major domain composed of an α-helical bundle-like structure. With the exception of Psf1, other subunits each has a small domain containing a three-stranded β-sheet core. Each full-length protein in the crystal has unstructured regions that are all located on the surface of GINS and are probably involved in its interaction with other replication factors. The four subunits contact each other mainly through α-helices to form a ring-like tetramer with a central pore. This pore is partially plugged by a 16-residue peptide from the Psf3 N-terminus which is unique to some eukaryotic Psf3 proteins and is not required for tetramer formation. Removal of this N-terminal 16 residues of Psf3 from the GINS tetramer increases the opening of the pore by 80%, suggesting a mechanism by which accessibility to the pore may be regulated. The structural data presented here indicate that the GINS tetramer is a highly stable complex with multiple flexible surface regions.; For the helicase part, I attempted to understand how LTag helicase assembles on the origin, initiates origin melting and DNA unwinding by determining the structure of a dimeric LTag bound to the EP half of the SV40 viral replication origin. This is the first LTag structure with both the origin binding domain (OBD) and the helicase domain visible and shows that the linker region between OBD and helicase domain is very flexible. The structural information reveals how a dimeric LTag recognizes and assembles on the replication origin: LTag is brought to the viral DNA by OBD recognizing its minimal binding sequence 5’-GC-3’. Once a LTag monomer encounters its canonical penta-nucleotide recognition sequence, it is locked onto the replication origin. The subsequent recruitment of the second LTag molecule to the same half of the origin is mainly mediated by protein-protein interactions between the two LTag subunits. This dimeric structure bound to the origin may also suggest a new mechanism of origin melting: DNA bending induced by LTag hexamer assembly at the replication origin at an angle to the global directionality of DNA creates torsional stress that is released by origin melting. The LTag Zn domain forms part of the LTag hexameric central channel and thus may play an important role in DNA bending and origin melting. It is also important for anchoring LTag hexamer to the replication origin by encircling dsDNA. The models proposed here for LTag assembly at the replication origin and origin melting may have general implications to other eukaryotic counterparts. |
| Keyword | DNA replication; GINS origin; helicase; SV40 large T antigen |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m3324 |
| Rights | Chang, Yu-Hao Paul |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-CHANG-3999 |
| Archival file | uscthesesreloadpub_Volume29/etd-CHANG-3999.pdf |
Description
| Title | Page 1 |
| Full text | STRUCTURAL STUDIES OF TWO KEY FACTORS FOR DNA REPLICATION IN EUKARYOTIC CELLS by Yu-Hao Paul Chang A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirement for the Degree DOCTOR OF PHILOSOPHY (GENETIC, MOLECULAR AND CELLULAR BIOLOGY) August 2010 Copyright 2010 Yu-Hao Paul Chang |
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