Date of Award

12-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Biochemistry

Committee Member

Dr. Michael Sehorn, Committee Chair

Committee Member

Dr. William Marcotte

Committee Member

Dr. Alex Feltus

Committee Member

Dr. Kerry Smith

Abstract

DNA double-strand breaks (DSBs) are the most threatening type of DNA damage in a cell. Homologous recombination (HR) is the most accurate repair mechanism for DSBs, and if HR fails, the integrity of the genome can be compromised. Two recombinases, RAD51 and DMC1, are vital for HR but require assistance for HR to proceed efficiently and accurately. Several proteins, including mediators, single-strand binding proteins, and accessory proteins, have been shown to function in the HR with the recombinases. Mediators are responsible for overcoming inhibition caused by the single-strand binding protein, Replication protein A (RPA). Accessory proteins assist the recombinases through DSB localization, ATP hydrolysis, filament stabilization and several other functions. In addition to RPA, higher eukaryotes possess two other SSBs, SSB1 and SSB2. Both hSSBs maintain genomic integrity through participation in the HR pathway. It was previously demonstrated that hSSB1 stimulates RAD51 during D-loop formation. Additionally, the hSSBs maintain genomic integrity through the repair of stalled replication forks. In this dissertation, we present in Chapter 2 surprising activities of the hSSBs that support the recent genetic data implicating hSSB1 and hSSB2 in the repair of stalled replication forks. We demonstrated a functional interaction with the human polymerase η in D-loop extension and second-end capture. This is the first report of the hSSBs interaction with a polymerase and identifies a new function of the hSSBs in DNA double-strand break repair. We also report that hSSB1 and hSSB2 can anneal single-strand DNA and melt double-strand DNA. In Chapter 3, we examined the effect of CaCl2 and MgCl2 on hSSB D-loop formation and demonstrate that hSSB1 and hSSB2 can in fact form D-loops in the absence of the recombinase, RAD51. Both hSSB1 and hSSB2 form a heterotrimeric complex with Integrator subunit 3 (INTS3) and the Single-strand interacting protein 1 (hSSBIP1). We have purified the components and confirmed complex formation. The effect of the complex proteins on D-loop extension by hPol η will be interesting to examine in the future. The hMEI5-SWI5 ortholog in Saccharomyces cerevisiae functions as a mediator to scDMC1. To date, there have been no reports regarding hMEI5-SWI5 functionality with hDMC1. In Chapter 3, we examined the DNA binding activity of hMEI5 and hSWI5 individually and as a complex (Mei5-Swi5), in addition to demonstrating physical interaction with both DMC1 and RPA. Importantly, we report that hMEI5 but not hSWI5 retains mediator activity to hDMC1 using an in vitro homologous DNA pairing assay. This is the first biochemical report on hMEI5-hSWI5.

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