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,Paul Russell,Eva Mejia-Ramirez,Oliver Limbo,Gregory P Copenhaver

doi:10.1371/journal.pgen.1002271

Petra Langerak, Paul Russell + Show 3 more

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https://doi.org/10.1371/journal.pgen.1002271

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Journal: PLoS Genetics	Publication Date: Sep 8, 2011
Citations: 282	License type: CC BY 4.0

Affiliation: Scripps Research Institute

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.

Highlights

Genome integrity is constantly threatened by DNA damage resulting from internal and external insults
In this study we have investigated the roles of Mre11 endonuclease activity and Ctp1 in DNA end resection and double-strand breaks (DSBs) repair
Our goal was to understand why these activities are crucial for DSB repair in fission yeast, and why the requirement for their critical functions can be bypassed by eliminating the Ku complex

Summary

Introduction

Genome integrity is constantly threatened by DNA damage resulting from internal and external insults. There are two major DSB repair pathways: nonhomologous end-joining (NHEJ) and homologous recombination (HR). NHEJ is an error-prone pathway that repairs DSBs by directly ligating DNA ends with little or no end processing. Key NHEJ proteins that are conserved from yeast to humans include the Ku70-Ku80 heterodimer, which binds DNA ends with high affinity, as well as XLF/Cernunnos and DNA ligase IV [2,3,4,5]. A multitude of human disease syndromes have been traced to NHEJ and HR defects, including those characterized by neurological, immunological and developmental disorders as well as radiation sensitivity, premature aging diseases and cancer, pointing to the critical roles of NHEJ and HR in maintaining genome stability [8,9,10,11,12,13]

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