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MECHANISM STUDY OF SV40 LARGE
TUMOR ANTIGEN ATPASE AND
HELICASE FUNCTIONS IN VIRAL DNA
REPLICATION
by
Xian Jessica Yu
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 PHILOSOPY
(MOLECULAR BIOLOGY)
December 2012
Copyright 2012 Xian Jessica Yu
Object Description
| Title | Mechanism study of SV40 large tumor antigen atpase and helicase functions in viral DNA replication |
| Author | Yu, Xian Jessica |
| Author email | xiany@usc.edu;yuxian66@hotmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Molecular Biology |
| School | College of Letters, Arts And Sciences |
| Date defended/completed | 2012-10-17 |
| Date submitted | 2012-11-16 |
| Date approved | 2012-11-16 |
| Restricted until | 2012-11-16 |
| Date published | 2012-11-16 |
| Advisor (committee chair) | Chen, Xiaojiang S. |
| Advisor (committee member) |
Forsburg, Susan Harworth, Ian |
| Abstract | Simian Virus 40 (SV40) has been studied as the model system to elucidate the mechanism of eukaryotic DNA replication. The oncogenic large tumor antigen (LTag) encoded by SV40 not only transforms cells and induces tumors but also functions as a molecular motor machine that melts the viral origin and unwinds duplex DNA to initiate replication. It’s been regarded as the functional homologue of minichromosome maintenance (MCM) protein, a putative replicative helicase in eukaryotic and archaeal cells. ❧ Simian virus 40 large tumor antigen (LTag) is an AAA+ hexameric motor that harnesses the energy from ATP binding/hydrolysis to initiate DNA replication and unwind replication forks. However, how the six subunits of LTag hexamer motor coordinate for ATP hydrolysis and for DNA unwinding/translocation are unresolved. Here I investigated the subunit coordination mechanisms for ATP hydrolysis and DNA unwinding through a series of mutant doping experiments. For ATP hydrolysis, I observed a random mode in the absence of DNA, a semi-random mode with ssDNA, and a semi-coordinated mode with fork or origin DNA. For DNA unwinding, however, the results indicated a semi-coordinated mode for fork-DNA, but a fully coordinated mode for origin DNA. These results and previous evidence suggest a distinctive coordination behavior for LTag, which adopts different coordination for ssDNA translocation, fork- DNA unwinding, and origin DNA unwinding. For origin DNA unwinding, LTag hexamer operates in a fully coordinated mode. ❧ The β-hairpin and F-loop in the central channel of LTag hexamer has been identified to play a key role in interacting with DNA substrate. Here, I made a series of mutations on the tip of β-hairpin and an adjacent F-loop structure. A variety of functional assays have been performed using these mutants. I demonstrate that two neighboring β- hairpin tip residues (K512 and H513) and residue F459 on F-loop are involved in DNA- dependent ATPase stimulation, in local origin melting, ss- and ds- DNA translocation, and fork- and origin- DNA unwinding. However, their relative functional roles in these activities are different, with K512 as a sensor to recognize and convey the DNA signal from the central channel while H513 and F459 as mechanics to interact with DNA base pairs for melting and unwinding. The biochemical results support the structural observations that the positively charged residue K512 interacts with DNA phosphate backbone through electrostatic interactions while ring-shaped residues H513 and F459 interact with DNA by hydrophobic stacking. ❧ In summary, my thesis is focused on the functional study of LTag in regard to ATP hydrolysis and DNA unwinding, as well as the role defining of important residues on β-hairpin and loop structure. The overall work will provide important clues towards studying other motor helicases in the same family and insights into the big picture of DNA initiation and replication. |
| Keyword | hexameric helicase; large T antigen; subunit coordination mechanism; SV40 replication |
| 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-m |
| Rights | Yu, Xian Jessica |
| 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 author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume4/etd-YuXianJess-1299-0.pdf |
Description
| Title | Page 1 |
| Full text | MECHANISM STUDY OF SV40 LARGE TUMOR ANTIGEN ATPASE AND HELICASE FUNCTIONS IN VIRAL DNA REPLICATION by Xian Jessica Yu 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 PHILOSOPY (MOLECULAR BIOLOGY) December 2012 Copyright 2012 Xian Jessica Yu |
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