Title: Replication Protein A in the Maintenance of Genome Stability
Description: High fidelity double strand break repair is paramount for the maintenance of genome integrity and faithful passage of genetic information to the following generation. Homologous recombination (HR) and non-homologous end joining (C-NHEJ) have evolved as the two major pathways for the efficient and accurate repair of double strand breaks (DSBs). In addition, a minor Ku- and Ligase IV-independent end-joining pathway has been identified and implicated in the formation of chromosomal translocations. This alternative end-joining pathway occurs by bridging the break ends through annealing between short microhomologies, hence the name microhomology-mediated end joining (MMEJ). In addition to these defined DSB repair pathways, a broken DNA end possesses immense mutagenic potential to generate chromosomal rearrangements. Diverse and complex rearrangements are a commonly observed feature amongst cancer cells. The focus of this thesis is to examine the role of Replication Protein A (RPA) in binding single-stranded DNA (ssDNA) repair intermediates to promote error free repair and to prevent mutagenic chromosomal deletions and rearrangements.

RPA is a highly conserved, heterotrimeric ssDNA binding protein with a ubiquitous role in all DNA transactions involving ssDNA intermediates. RPA promotes resection at DSBs to facilitate HR and abrogation of this function has severe consequences. Defective RPA can lead to the formation of secondary structures and impair loading of homology search proteins such as Rad52 and Rad51. Using a chromosomal end-joining assay, we demonstrate that hypomorphic rfa1 mutants exhibit elevated frequencies of MMEJ by up to 350-fold. Biochemical characterization of RPAt33 and RPAt48 complexes show these mutants are compromised for their ability to prevent spontaneous annealing and the removal of secondary structures to fully extend ssDNA. These results demonstrate that annealing between MHs defines a critical control to regulate MMEJ repair. Therefore, RPA bound to ssDNA intermediates shields complementary sequences from annealing to promote error-free HR and prevents repair by mutagenic MMEJ, thereby preserving genomic integrity.

RPA also impedes intrastrand annealing between short inverted repeat sequences to prevent the formation of foldback structures. Foldbacks have been proposed to drive palindromic gene amplification, a genome destabilizing rearrangement that can disrupt the protein expression equilibrium and is a prevalent phenomenon within tumor cells. Palindromic duplications are elevated ~1000-fold in rfa1-t33 sae2Δ and rfa1-t33 mre11-H125N mutants compared to sae2Δ or mre11-H125N, yet we did not detect these events in the hypomorphic rfa1-t33 mutant. This suggests that Mre11 and Sae2 play critical roles in preventing palindromic amplification through regulation of the Mre11 structure-specific endonuclease to process DNA foldbacks (also called DNA hairpins). Therefore, Mre11-Sae2 together with RPA prevent palindromic gene amplification. Together, these data focus the spotlight on RPA playing active central and supporting roles to sustain genome stability. This additionally raises that notion that secondary structures are potent instigators and mediators of many genome rearrangements and their prevention by RPA is absolutely crucial.