Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Acid Anhydride Hydrolases
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cell Cycle Proteins / physiology
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DNA Breaks, Double-Stranded*
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DNA Breaks, Single-Stranded
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DNA Helicases / genetics
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DNA Helicases / metabolism
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DNA Helicases / physiology
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DNA Repair / genetics*
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DNA Repair Enzymes / genetics
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DNA Repair Enzymes / metabolism
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DNA Repair Enzymes / physiology
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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DNA-Binding Proteins / physiology
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Exodeoxyribonucleases / genetics
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Exodeoxyribonucleases / metabolism
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Exodeoxyribonucleases / physiology
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Humans
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In Vitro Techniques
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MRE11 Homologue Protein
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Models, Biological
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Multiprotein Complexes / genetics
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Multiprotein Complexes / metabolism
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Multiprotein Complexes / physiology
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Nuclear Proteins / physiology
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Protein Binding / physiology
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RecQ Helicases / genetics
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RecQ Helicases / metabolism
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RecQ Helicases / physiology
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Replication Protein A / genetics
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Replication Protein A / metabolism
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Replication Protein A / physiology
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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MRE11 protein, human
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Multiprotein Complexes
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NBN protein, human
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Nuclear Proteins
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Replication Protein A
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Exodeoxyribonucleases
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MRE11 Homologue Protein
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exodeoxyribonuclease I
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Acid Anhydride Hydrolases
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RAD50 protein, human
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Bloom syndrome protein
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DNA Helicases
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DNA2 protein, human
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RecQ Helicases
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DNA Repair Enzymes