Abstract
Homologous recombination (HR) and translesion synthesis (TLS) promote gap-filling DNA synthesis to complete genome replication. One factor involved in both pathways is RAD18, an E3 ubiquitin ligase. Although RAD18's role in promoting TLS through the ubiquitination of PCNA at lysine 164 (K164) is well established, its requirement for HR-based mechanisms is currently less clear. To assess this, we inactivated RAD18 in three human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e . HCT116 mutants were hyper-recombinogenic (hyper-rec). Loss of RAD18 also impaired TLS activity in HCT116 cells, but unexpectedly, did not reduce clonogenic survival. Interestingly, these phenotypes appear linked to PCNA K164 ubiquitination, as HCT116 PCNA (K164R/+) mutants were also hyper-rec and showed reduced TLS activity, consistent with previous studies in rad18 (-/-) or pcna (K164R) avian DT40 mutant cells. Importantly, knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. Taken together, these data suggest that the roles of human RAD18 in directing distinct gap-filling DNA synthesis pathways varies depending on cell type and that these functions are linked to PCNA ubiquitination.