release of ku and mrn from dna ends by mre11 nuclease activity and ctp1 is required for homologous recombination repair of double-strand breaks从dna末端释放ku和mrn mre11核酸酶活性和ctp1同源重组修复需要的双链断裂.pdfVIP

Release of Ku and MRN from DNA Ends by Mre11 Nuclease Activity and Ctp1 Is Required for Homologous Recombination Repair of Double-Strand Breaks Petra Langerak, Eva Mejia-Ramirez, Oliver Limbo, Paul Russell* Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, United States of America 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 59-to-39 resection of DNA ends, generating 39 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