Ngo, Greg H. P. and Balakrishnan, Lata and Dubarry, Marion and Campbell, Judith L. and Lydall, David (2014) The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1. Nucleic Acids Research, 42 (16). pp. 10516-10528. ISSN 0305-1048. http://resolver.caltech.edu/CaltechAUTHORS:20140820-085638030
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Single-stranded DNA (ssDNA) at DNA ends is an important regulator of the DNA damage response. Resection, the generation of ssDNA, affects DNA damage checkpoint activation, DNA repair pathway choice, ssDNA-associated mutation and replication fork stability. In eukaryotes, extensive DNA resection requires the nuclease Exo1 and nuclease/helicase pair: Dna2 and Sgs1^(BLM). How Exo1 and Dna2-Sgs1^(BLM) coordinate during resection remains poorly understood. The DNA damage checkpoint clamp (the 9-1-1 complex) has been reported to play an important role in stimulating resection but the exact mechanism remains unclear. Here we show that the human 9-1-1 complex enhances the cleavage of DNA by both DNA2 and EXO1 in vitro, showing that the resection-stimulatory role of the 9-1-1 complex is direct. We also show that in Saccharomyces cerevisiae, the 9-1-1 complex promotes both Dna2-Sgs1 and Exo1-dependent resection in response to uncapped telomeres. Our results suggest that the 9-1-1 complex facilitates resection by recruiting both Dna2-Sgs1 and Exo1 to sites of resection. This activity of the 9-1-1 complex in supporting resection is strongly inhibited by the checkpoint adaptor Rad9^(53BP1). Our results provide important mechanistic insights into how DNA resection is regulated by checkpoint proteins and have implications for genome stability in eukaryotes.
|Additional Information:||© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received June 18, 2014; Revised July 17, 2014; Accepted August 1, 2014. First published online: August 13, 2014. We thank Mike Boxem and Matija Dreze for the generous gifts of strains and plasmids; Aziz Sancar, Robert Bambara, Laura Lindsey-Boltz and Paul Modrich for generously providing purified proteins and advice; and members of the Lydall and Campbell labs for comments on the manuscript. Funding: Wellcome Trust [075294, 093088 to D.L]; National Institutes of Health [GM098328 to L.B.; GM100186 to J.L.C.]; European Molecular Biology Organization [EMBO ALTF218-2012 to M.D.]. Funding for open access charge: Wellcome Trust [075294, 093088]. Conflict of interest statement. None declared.|
|Official Citation:||Greg H.P. Ngo, Lata Balakrishnan, Marion Dubarry, Judith L. Campbell, and David Lydall The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1 Nucl. Acids Res. (15 September 2014) 42 (16): 10516-10528 first published online August 13, 2014 doi:10.1093/nar/gku746 Acids Research. doi: 10.1093/nar/gku746|
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|Deposited By:||Jason Perez|
|Deposited On:||20 Aug 2014 18:07|
|Last Modified:||11 Feb 2015 17:29|
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