Homologous recombination plays a critical role in genome stability by repairing DNA double strand breaks (DSBs) and rescuing stalled replication forks. RAD51 catalyzes the central reactions of homologous recombination: homology search and strand invasion. RAD51-/- mutant mice die early in embryogenesis and cells depleted of RAD51 accumulate DSBs prior to cell death. Due to the central role of RAD51 in homologous recombination, this led to the prevailing view that the essential function of RAD51 is the homologous recombination-mediated repair of DSBs that occur spontaneously during replication. However, recent studies have identified recombination-independent roles for RAD51 and other homologous recombination factors including the breast cancer susceptibility proteins, BRCA1 and BRCA2, in replication fork stability. Previously, we generated a separation-of-function allele of RAD51 in budding yeast that retains the ability to form a nucleoprotein filament on single-stranded DNA, but is defective in homology search and strand invasion (rad51-II3A). Surprisingly, this Rad51 mutant did not exhibit the growth defect observed in Rad51-deficient yeast cells, indicating the growth defect in yeast is not a result of failure to repair DSBs. This result combined with the recent evidence that RAD51 has recombination-independent roles in replication led us to the hypothesis that the essential function of RAD51 in vertebrate cells is replication fork stability, and not homologous recombination. Using Cas9/CRISPR, we will generate derivatives of human cell lines that express RAD51-II3A protein from the RAD51 genomic locus. Using these cell lines, we will determine the effect of loss of strand exchange activity on cell viability and chromosome stability in unperturbed cells. Finally, we will examine the response of RAD51-II3A expressing cells to replication stress and to genotoxic agents that induce DNA double strand breaks. We will determine if the RAD51-II3A cells have the ability to protect replication forks and restart in response to replication perturbation, but lack the abilityto repair DSBs. The proposed work will provide valuable insight to the HR-independent functions of RAD51 in cell viability and replication fork stability. Given the importance of HR in protecting cells from genome instability, this work will provide important mechanistic insight into how disruption of HR results in predisposition to cancer.
We propose to engineer tumor cells that express a mutant form of the central homologous recombination factor, RAD51, which retains the ability to function during replication, but is unable to promote homologous recombination. This mutant will allow us to determine if the essential function of RAD51 involves its role in homologous recombination and also provide a key tool for understanding both recombination-dependent and recombination-independent functions of RAD51. These studies will lead to a better understanding on how recombination proteins promote genome stability and prevent cancer.