Functional redundancy between the RAD51 accessory proteins RAD51AP1 and RAD54 in homologous recombination DNA repair

2021 Spring.Includes bibliographical references.Cancer is responsible for the death of millions of people annually. Factors that increase the risk of tumorigenesis are endogenous challenges and exogenous compounds. These insults are responsible for the generation of DNA lesions, the most toxic one of which is a DNA double-strand break (DSB). DSBs can be repaired by several different DNA repair pathways, among which homologous recombination (HR) is the least error prone. In HR, DNA strand exchange is mediated by the RAD51 recombinase which forms a nucleoprotein filament on single-stranded DNA for strand invasion. RAD51-mediated strand invasion is supported by the DNA motor protein RAD54 and by the RAD51-Associated Protein 1 (RAD51AP1). While the pre-synaptic steps of HR in human cells have been studied extensively, there are still extensive knowledge gaps with respect to the molecular mechanisms of synapsis and post-synapsis and the roles of RAD51AP1 and RAD54 in these later steps of HR. Here, I hypothesized that RAD51AP1 and RAD54 may exhibit functional redundancy in human cells. Also, I speculated that Rad51ap1 disruption in mice would be associated with an increased susceptibility of these mice to radiation carcinogenesis. Finally, I hypothesized that post-translational modification and, more specifically, phosphorylation may regulate the activity of human RAD51AP1. To test for functional redundancy between RAD51AP1 and RAD54, we investigated the impact of simultaneous RAD51AP1 and RAD54 disruption in human cancer cell lines and in response to DNA-damaging agents in cell survival and DNA replication assays. We found that cells lacking both RAD51AP1 and RAD54 (i.e., double KO cells) are more sensitive to the cytotoxic effects of mitomycin C (MMC) or olaparib exposure than cells lacking either RAD51AP1 or RAD54. Accordingly, double KO cells exhibit a more pronounced G2/M arrest, higher levels of chromosomal aberrations and increased sensitivity to DNA replication stress as determined by DNA combing experiments. These results show that RAD51AP1 and RAD54 can compensate for each other in human cancer cell lines. To investigate the consequences of RAD51AP1 loss in mice we utilized a novel mouse model that lacks Rad51ap1 and determined the susceptibility of these mice to radiation carcinogenesis. We found that compared to wild type mice, loss of Rad51ap1 does not affect the survival of mice after whole body IR. We speculate that functional redundancy between RAD51AP1 and RAD54 may also exist in mice, and that Rad51ap1-/- Rad54-/- double KO mice may exhibit pronounced susceptibility to radiation carcinogenesis. Finally, we sought to characterize the regulation of RAD51AP1 activity by post-translational modification. To achieve this objective, we identified two critical residues in RAD51AP1 that appear to be regulated by phosphorylation, S277 and S282. We found that mutation of these residues to the non-phosphorylatable S277A and S282A compromises RAD51AP1 function as measured by DNA replication and cell survival assays. These results suggest that phosphorylation of S277 and/or S282 is crucial for RAD51AP1 function. Collectively, our studies clarify one aspect of functional redundancy within the HR pathway, and the role of post-translational modification of RAD51AP1. Our results provide new insights on the mild phenotypes associated with RAD51AP1 or RAD54 deficiency in human cells and mice. Our findings highlight the importance of development of personalized approaches for cancer treatment