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Nuclease Assays with pN01 from TopoGEN

Nuclease Assays with pN01 from TopoGEN

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

Eun Yong Shim1,4, Woo-Hyun Chung2,4, Matthew L Nicolette3, Yu Zhang1, Melody Davis1, Zhu Zhu2, Tanya T Paull3, Grzegorz Ira2,* and Sang Eun Lee1,* 1Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 2Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA and 3Department of Molecular Genetics and Microbiology, The Howard Hughes Medical Institute, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA

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 50-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.

The EMBO Journal (2010) 29, 3370–3380. doi:10.1038/
emboj.2010.219; Published online 10 September 2010
Subject Categories: genome stability & dynamics
Keywords: double-strand break; Ku; Mre11; resection;
Saccharomyces cerevisiae

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