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Published February 15, 2011 | Published + Supplemental Material
Journal Article Open

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


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.

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.

Attached Files

Published - Nimonkar2011p12977Genes_Dev.pdf

Supplemental Material - NimonkarSuppMat.pdf


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