Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks

Single‐stranded DNA constitutes an important early intermediate for homologous recombination and damage‐induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double‐strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in...

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Bibliographic Details
Published in:The EMBO journal 2010-10, Vol.29 (19), p.3370-3380
Main Authors: Shim, Eun Yong, Chung, Woo-Hyun, Nicolette, Matthew L, Zhang, Yu, Davis, Melody, Zhu, Zhu, Paull, Tanya T, Ira, Grzegorz, Lee, Sang Eun
Format: Article
Language:English
Subjects:
Chromatin Immunoprecipitation
Deoxyribonucleic acid
DNA
DNA Breaks, Double-Stranded
DNA Helicases - metabolism
DNA Repair
DNA-Binding Proteins - metabolism
double-strand break
Endodeoxyribonucleases - metabolism
Endonucleases - metabolism
Enzymes
Exodeoxyribonucleases - metabolism
Mre11
Multiprotein Complexes - metabolism
Proteins
resection
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins - metabolism
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Summary:Single‐stranded DNA constitutes an important early intermediate for homologous recombination and damage‐induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double‐strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5′‐strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2‐dependent initiation to the Exo1‐ and Dna2‐dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation. Defined biochemical and genetic assays show that the MRX complex promotes recruitment of resection nuclease in part by counteracting Ku binding at double strand breaks, providing insight into the switch from initial end processing to processive long‐range resection during homologous recombination repair.
ISSN:0261-4189
1460-2075
DOI:10.1038/emboj.2010.219