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A Study of DNA Homologous Recombination Mechanism through Biochemical Characterization of Rad52 and BRCA2 in Yeast and Humans

Khade, Nilesh V.
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1437664889

Abstract Details

2015, Doctor of Philosophy (PhD), Ohio University, Biological Sciences (Arts and Sciences).
Homologous recombination (HR) is critical for double strand break repair and maintenance of genome stability. The recombination mediator proteins in HR play key roles by recruiting RecA-family recombinases like Rad51 to single-stranded DNA. In vitro, mediator proteins bind to their cognate Rad51 recombinases and mediate their loading onto single-stranded DNA which catalyses homology search and DNA strand invasion, a unique and essential process in HR. In yeast, the mediator function is characteristic of the Rad52 (yRad52) protein which also promotes ssDNA annealing. In humans, the breast cancer susceptibility protein (BRCA2) possess the mediator function while the human Rad52 protein is thought to anneal complementary single stranded DNA. However, the role of human Rad52 (hRad52) in HR has remained elusive and no in vitro mediator activity of human Rad52 has been reported. In this study, the loss of mediator function in hRad52 was investigated by examining interspecies interactions between the yeast and human mediators with human Rad51 (hRad51) recombinase. Interestingly, yRad52 successfully mediated loading of hRad51 recombinase onto the single-stranded DNA complexed with yeast RPA in vitro. This recombinase-loading activity was abolished when yeast RPA (E.coli SSB homolog in yeast) was replaced with human RPA, suggesting that species-specific interaction between Rad52 and RPA is important for mediator activity. In this study, the hRad51 binding domain was identified in the yRad52 C-terminus; deletion of the C-terminus (yRad52NM) resulted in loss of mediator function. hRad52 was also tested for mediator activity but it failed to mediate loading of hRad51 in presence of either hRPA or yRPA. As yeast and human Rad52 share similarity between their N-terminal domains, the C-terminal Rad51-binding domain of human Rad52 was replaced with BRC repeats of BRCA2, which is known to interact with hRad51. In another experiment, the hRad52 C-terminal domain was replaced with the yRad52 C-terminal region. Unexpectedly, hRad52-BRC4 and hRad52NM-y52C fusions did not attain mediator activity for human Rad51. Mediator function was not present even when the yRad52 C-terminal domain was replaced with either multiple BRC4 repeats or the BRC3-BRC4 repeat of BRCA2. Taken together, these results indicate that BRC repeats cannot substitute the function of the C-terminal Rad51-binding domain of human or yeast Rad52 and furthermore, an intact yRad52 is required for hRad51 mediator function. In addition, the yRad52 C-terminal domain was determined to be required for efficient ssDNA annealing.
Tomohiko Sugiyama (Advisor)
Donald Holzschu (Committee Member)
Alan Showalter (Committee Member)
Frank Horodyski (Committee Member)
173 p.
Biochemistry; Biology
DNA Repair; Double Strand Break; Homologous Recombination; Mediator Activity; Annealing Activity; Rad52; BRCA2

Recommended Citations

Citations

  • Khade, N. V. (2015). A Study of DNA Homologous Recombination Mechanism through Biochemical Characterization of Rad52 and BRCA2 in Yeast and Humans [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1437664889

    APA Style (7th edition)

  • Khade, Nilesh. A Study of DNA Homologous Recombination Mechanism through Biochemical Characterization of Rad52 and BRCA2 in Yeast and Humans. 2015. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1437664889.

    MLA Style (8th edition)

  • Khade, Nilesh. "A Study of DNA Homologous Recombination Mechanism through Biochemical Characterization of Rad52 and BRCA2 in Yeast and Humans." Doctoral dissertation, Ohio University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1437664889

    Chicago Manual of Style (17th edition)

ohiou1437664889
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© 2015, all rights reserved.
This open access ETD is published by Ohio University and OhioLINK.