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Multi-disciplinary Investigation of the Kinetics and Protein Conformational Dynamics of DNA Replication and Oxidative DNA Damage Bypass and Repair

Maxwell, Brian Andrew
http://rave.ohiolink.edu/etdc/view?acc_num=osu1405961617

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Biophysics.
Genomic DNA is under constant attack from both environmental sources as well as toxic byproducts of natural cellular processes. Cells therefore must utilize a variety of pathways to repair or otherwise cope with damaged DNA in order to survive and to faithfully copy the genome during cell division. Unrepaired DNA damage sites or “lesions” can act as roadblocks to DNA replication by stalling the cell’s replicative DNA polymerase, a molecular motor responsible for copying the genome. In order to avoid cell death due to stalled DNA replication, DNA lesions can be bypassed through DNA translesion synthesis (TLS) whereby a stalled high-fidelity replicative DNA polymerase is replaced by a specialized lesion-bypass DNA polymerase capable of bypassing the damaged site. However, these specialized DNA lesion-bypass polymerases are much more error prone during DNA replication than replicative DNA polymerases, and mistakes made in copying the DNA during this process can potentially lead to cancer formation. Therefore polymerase switching during TLS must be tightly regulated to minimize the introduction of mutations while still allowing for TLS. In addition, cells also utilize various enzymes in the Base Excision Repair (BER) pathway to remove DNA lesions prior to the replication of DNA in order to avoid the potential mistakes that could be made during TLS. The goal of my research has been to establish an in depth understanding of the molecular mechanisms of the replication of damaged and undamaged DNA and of the BER pathway using a variety of in vitro biochemical and biophysical tools. My working hypothesize is that, in order to regulate polymerase switching and DNA damage repair, DNA damage sites can alter various properties of DNA polymerases and the enzymes involved in BER including how they bind to DNA, the rate at which they perform their catalytic functions and how they change their structures during the course of the reaction. In order to test this hypothesis I have utilized rapid mixing techniques to investigate how the presence of a DNA lesion will affect the rates of DNA copying for both replicative and specialized DNA polymerases. By utilizing fluorescence spectroscopy I have investigated how the presence of DNA damage affects the structural changes that the enzymes undergo when binding to DNA as well as when interacting with other substrates and protein cofactors. These fluorescence based studies make use of Forster Resonance Energy Trans (FRET), which serves as a “molecular ruler” to monitor the change in distance between two fluorescent markers which can be placed at specific locations on the enzymes and/or DNA. Finally, I have investigated the interactions between the enzymes and the DNA or between two enzymes on the level of individual molecules using a state-of-the-art single molecule FRET microscopy system that I designed and assembled. Collectively, the results from this research project have provided novel insight into the conformational dynamics and kinetics of the molecular interactions involved in DNA replication and DNA damage repair in the BER pathway.
Zucai Suo, PhD (Advisor)
Ralf Bundschuh, PhD (Committee Member)
Richard Swenson, PhD (Committee Member)
346 p.
Biophysics
DNA polymerase, FRET, Fluorescence, Single-molecule, TIRF, glycosylase, enzymology, kinetics, stopped-flow, conformational dynamics, Lesion bypass, DNA damabe repair, pre-steady state kinetics, Dpo4, base excision repair, DNA replication

Recommended Citations

Citations

  • Maxwell, B. A. (2014). Multi-disciplinary Investigation of the Kinetics and Protein Conformational Dynamics of DNA Replication and Oxidative DNA Damage Bypass and Repair [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1405961617

    APA Style (7th edition)

  • Maxwell, Brian. Multi-disciplinary Investigation of the Kinetics and Protein Conformational Dynamics of DNA Replication and Oxidative DNA Damage Bypass and Repair . 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1405961617.

    MLA Style (8th edition)

  • Maxwell, Brian. "Multi-disciplinary Investigation of the Kinetics and Protein Conformational Dynamics of DNA Replication and Oxidative DNA Damage Bypass and Repair ." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1405961617

    Chicago Manual of Style (17th edition)

osu1405961617
605
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This open access ETD is published by The Ohio State University and OhioLINK.