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Insights into Regulation of Human RAD51 Nucleoprotein Filament Activity During Homologous Recombination

Amunugama, Ravindra Bandara
http://rave.ohiolink.edu/etdc/view?acc_num=osu1321984760

Abstract Details

2011, Doctor of Philosophy, Ohio State University, Biophysics.
Homologous recombination (HR) is a mechanistically conserved pathway that occurs during meiosis and following the formation of DNA double strand breaks (DSBs) induced by exogenous stresses such as ionization radiation. HR is also involved in restoring replication when replication forks have stalled or collapsed. Defective recombination machinery leads to chromosomal instability and predisposition to tumorigenesis. However, unregulated HR repair system also leads to similar outcomes. Fortunately, eukaryotes have evolved elegant HR repair machinery with multiple mediators and regulatory inputs that largely ensures an appropriate outcome. A fundamental step in HR is the homology search and strand exchange catalyzed by the RAD51 recombinase. This process requires the formation of a nucleoprotein filament (NPF) on single-strand DNA (ssDNA). In Chapter 2 of this dissertation I describe work on identification of two residues of human RAD51 (HsRAD51) subunit interface, F129 in the Walker A box and H294 of the L2 ssDNA binding region that are essential residues for salt-induced recombinase activity. Mutation of F129 or H294 leads to loss or reduced DNA induced ATPase activity and formation of a non-functional NPF that eliminates recombinase activity. DNA binding studies indicate that these residues may be essential for sensing the ATP nucleotide for a functional NPF formation. An intriguing structural motif in the nucleotide-binding subunit interface of RAD51 is known as the ATP cap. The RAD51 family of recombinases contains a conserved aspartate that forms a salt bridge with the terminal phosphate of ATP, which is the likely source of a nonphysiological cation requirement for the formation of an active RAD51 NPF. In Chapter 3, I describe the biochemical analyses of a lysine substitution mutation of this conserved aspartate. The prototypical bacterial recombinase RecA as well as most RAD51 paralogs contains a conserved lysine at the analogous position. The HsRAD51(D316K) substitution mutant possess a reduced protein turnover from DNA that results in improved recombinase functions. Structural analyses indicates that HsRAD51(D316K) and its archaebacterial homolog form extended active NPF without the requirement of salt. These studies suggest that HsRAD51(D316) salt bridge may function as a conformational sensor that enhances RAD51 turnover at the expense of recombinase activity. In Chapter 4, I examination the role of the RAD51 paralog complex RAD51B-RAD51C as a potential mediator of RAD51 catalyzed D-loop formation. Modeled structures indicate that RAD51, RAD51B and RAD51C appear to have similar domain orientations within a NPF. Furthermore, RAD51B-RAD51C form stable complexes on ssDNA and partially stabilizes RAD51 NPF from the anti-recombinogenic activity of BLM. At sub-stoichiometric levels, RAD51B-RAD51C may modestly stimulate RAD51 mediated D-loop formation in presence of RPA. Collectively, these studies provide additional mechanistic and structural insight into the regulation of the RAD51 NPF during the homology search and strand exchange processes that is critical for efficient HR in human cells.
Richard Fishel, PhD (Advisor)
Jeffrey Parvin, MD PhD (Committee Member)
Charles Bell, PhD (Committee Member)
Michael Poirier, PhD (Committee Member)
Biochemistry; Biophysics; Molecular Biology
Homologous Recombination; DNA Repair; RAD51; RAD51 paralogs

Recommended Citations

Citations

  • Amunugama, R. B. (2011). Insights into Regulation of Human RAD51 Nucleoprotein Filament Activity During Homologous Recombination [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1321984760

    APA Style (7th edition)

  • Amunugama, Ravindra. Insights into Regulation of Human RAD51 Nucleoprotein Filament Activity During Homologous Recombination. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1321984760.

    MLA Style (8th edition)

  • Amunugama, Ravindra. "Insights into Regulation of Human RAD51 Nucleoprotein Filament Activity During Homologous Recombination." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1321984760

    Chicago Manual of Style (17th edition)

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© 2011, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.