Break dosage, cell cycle stage and DNA replication influence DNA double strand break response

Abstract

DNA double strand breaks (DSBs) can be repaired by non‐homologous end joining (NHEJ) or homology‐directed repair (HR). HR requires nucleolytic degradation of 5′ DNA ends to generate tracts of single‐stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae . We show that resection of an HO endonuclease‐induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3′ ssDNA tails. We show that both DSB resection and checkpoint activation are dose‐dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ‐independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights.