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THE CONTRIBUTION OF CHEMICAL STEPS TO DNA POLYMERASE BETA
CATALYSIS AND FIDELITY
by
Christopher Anthony Sucato
—————————————————————————————————
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL,
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
May 2008
Copyright 2008 Christopher Anthony Sucato
Object Description
| Title | The contribution of chemical steps to DNA polymerase beta catalysis and fidelity |
| Author | Sucato, Christopher Anthony |
| Author email | csucato@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Chemistry |
| School | College of Letters, Arts and Sciences |
| Date defended/completed | 2007-11-19 |
| Date submitted | 2008 |
| Restricted until | Unrestricted |
| Date published | 2008-03-03 |
| Advisor (committee chair) | Goodman, Myron |
| Advisor (committee member) |
Petruska, John McKenna, Charles Prakash, Surya |
| Abstract | The mechanism of DNA polymerase beta-catalyzed nucleotidyl transfer consists of chemical steps involving primer 3 OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. The nature of the rate-limiting step and active site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Using analogs of dGTP where the beta-gamma bridging oxygen is replaced with fluorine-substituted methylene groups the contribution of leaving-group elimination to the overall catalytic rate is examined. The analysis is then expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogs opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogs, dependent on the identity of the nascent base pair.; This observation, and the absence of an analogous effect on ground state dNTP binding (Kd values), points to active-site structural differences that become pronounced at the chemical transition state, and may possibly involve the conformation of the triphosphate moiety coordinated to Mg2+ or the orientation of Arg183 relative to the beta-gamma bridging oxygen. Catalytic differences with bulky halo-groups are manifested in the fidelity of T-G incorporation, where the CCl2-bridging analog shows a 25-fold increase in fidelity over the natural dGTP. This anti-mutator effect is notable since the CCl2-bridging leaving group is the bisphosphonate drug clodronate, well known for its antagonistic effect on bone tumor metastasis. Solvent pH and deuterium isotope-effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation. |
| Keyword | DNA polymerase; mechanistic enzymology; enzyme kinetics; base excision repair; replication fidelity; dNTP analogs |
| 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 |
| Type | texts |
| Legacy record ID | usctheses-m1035 |
| Rights | Sucato, Christopher Anthony |
| 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-Sucato-20080303 |
| Archival file | uscthesesreloadpub_Volume40/etd-Sucato-20080303.pdf |
Description
| Title | Page 1 |
| Full text | THE CONTRIBUTION OF CHEMICAL STEPS TO DNA POLYMERASE BETA CATALYSIS AND FIDELITY by Christopher Anthony Sucato ————————————————————————————————— A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL, UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) May 2008 Copyright 2008 Christopher Anthony Sucato |
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