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BLM–DNA2–RPA–MRN and EXO1–BLM–RPA–MRN constitute two DNA end resection machineries for human DNA break repair

Nimonkar, Amitabh V. and Genschel, Jochen and Kinoshita, Eri and Polaczek, Piotr and Campbell, Judith L. and Wyman, Claire and Modrich, Paul and Kowalczykowski, Stephen C. (2011) BLM–DNA2–RPA–MRN and EXO1–BLM–RPA–MRN constitute two DNA end resection machineries for human DNA break repair. Genes and Development, 25 (4). 350 -362. ISSN 0890-9369. PMCID PMC3042158.

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Repair of dsDNA breaks requires processing to produce 3′-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5′ → 3′ resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells.

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Additional Information:© 2011 Cold Spring Harbor Laboratory Press. Received October 17, 2010; revised version accepted January 4, 2011. We thank S.C.K. laboratory members for comments. We are grateful to Dr. David Chen (University of Texas Southwestern) for WRN, Dr. Ian Hickson (Oxford University) for RecQ5, Dr. Alex Mazin (Drexel University) for BLM helicase mutant (K695R), Dr. Patrick Sung (Yale University) for RecQ4, Petr Cejka (this laboratory) for Sgs1 and yDna2, Behzad Rad (this laboratory) for RecQ and SSB, Katsumi Morimatsu (this laboratory) for RecJ, and Zeynep Ozsoy (former laboratory member) for RPA. This work was supported by NIH grant GM-62653 to S.C.K., NCI program project SBDR 5PO1CA092548 and Netherlands Organization for Scientific Research-Chemical Sciences (NWO-CW) Vici award to C.W., Army Research Office grant ARO 09-1-0041 and NIH grant GM-78666 to J.L.C, and NIH grant GM-45190 to P.M. P.M. is an Investigator of the Howard Hughes Medical Institute.
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Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)UNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Subject Keywords:homologous recombination; DNA break repair; DNA end resection; BLM helicase; DNA2 nuclease; EXO1 nuclease
Issue or Number:4
PubMed Central ID:PMC3042158
Record Number:CaltechAUTHORS:20110316-161116107
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:22948
Deposited By: Jason Perez
Deposited On:17 Mar 2011 19:33
Last Modified:03 Oct 2019 02:41

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