Release of Ku and MRN from DNA Ends by Mre11 Nuclease Activity and Ctp1 Is Required for Homologous Recombination Repair of Double-Strand Breaks

Abstract

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5′-to-3′ resection of DNA ends, generating 3′ single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.

Author Summary

A double-strand break (DSB) is a devastating form of DNA damage. Fortunately, cells are equipped with two DSB repair pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ). The Mre11-Rad50-Nbs1 (MRN) protein complex recognizes DSBs and initiates HR repair. The Mre11 subunit harbors a nuclease domain that is essential for repair in fission yeast and mammals, although the function is unknown. Here we show that Mre11 nuclease activity is required to release the Ku complex from DNA ends in fission yeast. While the initiation of repair, i.e. the generation of single-stranded DNA (ssDNA) overhangs in Mre11-nuclease dead mutants, is unaffected, we find that an essential downstream step involving the localization of Replication Protein A (RPA) to ssDNA is substantially decreased due to the inability to release Ku and MRN from the DNA end. In contrast, a DNA processing factor called Ctp1, which binds to Nbs1, is essential for the initiation of repair as well as the release of Ku and MRN from DNA ends. Importantly, we find that efficient localization of RPA, which is essential for efficient DSB repair by HR, requires the release of Ku and MRN from the DNA by the combined action of Mre11 nuclease and Ctp1.