A Novel Role for Replication Protein A in Double-Strand Break Repair

Abstract

DNA encodes the instructions for all cellular processes. To survive and proliferate, cells must maintain and replicate their genomes in the face of chemical, biological and environmental DNA damaging agents. Pathways that repair DNA lesions are thus essential to cell survival, and dysregulation of these pathways is a frequent source of tumorigenesis. Among the diverse forms of DNA damage, none is thought to be more deleterious than double-strand breaks (DSBs). Despite decades of research devoted to understanding how cells sense and repair DSBs, the mechanisms underlying these pathways remain incompletely understood. One critical question is how error-prone DNA repair pathways compete with more accurate repair pathways to repair DSBs. The goal of my graduate studies was to better understand the molecular mechanism of the mutagenic repair pathway polymerase theta-mediated end-joining (TMEJ), which is upregulated in many cancers. Using advances in protein structure prediction, we screened genome maintenance proteins in silico for predicted interactions with a core mediator of TMEJ – the multifunctional enzyme polymerase theta (Polθ). We identified the heterotrimeric eukaryotic single-strand binding protein RPA as the top predicted interaction. While RPA has traditionally been thought to inhibit TMEJ based on partial depletion in cells, we show using a cell-free TMEJ assay in Xenopus egg extracts that its full depletion abolishes end joining. By dividing Polθ into segments and repeating our in silico screen, we identified four additional predicted interactions between the two proteins and validated their importance in extracts. We show that the N-terminus of RPA1 recruits Polθ via two interactions with conserved regions of the Polθ linker domain. We also show that RPA2 and the C-terminus of RPA1 promote TMEJ by stimulating the helicase domain of Polθ to simultaneously bind DNA, anneal two distinct strands and destabilize and remove RPA. These findings show how RPA, once thought to inhibit TMEJ, promotes repair. Taken together, they deepen our understanding of how Polθ repairs breaks and lay the foundation for further study into how this mutagenic repair pathway is selected over alternative DNA repair processes.