Mechanism and regulation of DNA end processing

Abstract

DNA ends exposed after introduction of double‐strand breaks (DSBs) undergo 5′‐3′ nucleolytic degradation to generate single‐stranded DNA (ssDNA), the substrate for binding by the Rad51 protein to initiate homologous recombination. We have shown that resection of DNA ends occurs by a two‐step process. In the first step, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. In a second step, Exo1 or Sgs1 with Dna2 rapidly process the minimally resected intermediate to generate long tracts of ssDNA that serve as substrate for Rad51. In the absence of Exo1 and Sgs1, the partially resected intermediates accumulate and are poor substrates for homology‐dependent repair (HDR). RPA plays an active role in resection by stimulating both Exo1 and Sgs1‐dependent end processing and controls the polarity of degradation. The initial processing step by the Mre11 complex and Sae2 is cell cycle regulated and appears to be important to promote HDR and to prevent repair by non‐homologous end joining. In the absence of end protection by Ku, we have shown that the requirement for the MRX complex is bypassed and Exo1 executes resection. In contrast, both the Exo1 and Sgs1 resection pathways contribute to DSB processing in the absence of Ku and Sae2 or when the MRX complex is intact but functionally compromised by elimination of the Mre11 nuclease activity. In addition, we show that replication associated DSBs need to be processed by Sae2 for repair by HR unless Ku is absent. Finally, we show that the presence of Ku or the MRX‐Tel1 damage checkpoint exacerbates DNA end‐processing defects established in the sae2Δ sgs1Δ mutant, leading to its lethality.