Saccharomyces cerevisiaeRev7 regulates DSB repair pathway choice through binding and blocking Mre11 nuclease and Rad50 ATPase activities

Recent studies in cancer cell lines have shown that the tetrameric Shieldin complex (comprising REV7, SHLD1, SHLD2, and SHLD3) facilitates non-homologous end-joining (NHEJ), while blocking homologous recombination (HR). Surprisingly, several eukaryotic species lack SHLD1, SHLD2 and SHLD3 orthologs, suggesting that Rev7 may leverage an alternative mechanism to regulate the double-strand break (DSB) repair pathway choice. Exploring this hypothesis, we discovered thatSaccharomyces cerevisiaeRev7 robustly interacts with the Mre11-Rad50-Xrs2 (MRX) subunits, impedes G-quadruplex DNA synergised, HU-induced toxicity and facilitates NHEJ, while antagonizing HR. We identified a 42-aminoacid C-terminal fragment of Rev7 that was critical for its binding to the subunits of MRX complex, protectrev7Δcells from G-quadruplex DNA-HU-induced toxicity and promote NHEJ by inhibiting HR, whereas the N-terminal HORMA domain, a conserved protein–protein interaction module, was dispensable. We further demonstrate that the full-length Rev7 impedes Mre11 nuclease and Rad50’s ATPase activities, without affecting the latter’s ATP-binding ability. Notably, we found that Rev7 binds with high affinity and specificity to G-quadruplex structures, as opposed to no binding to mixed-sequence single- and double-stranded DNA. These data uncover unanticipated insights into the functional interaction between the MRX subunits and Rev7, and highlight a mechanism by which it regulates the DSB repair pathway choice between HR and NHEJ inS. cerevisiae.