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快乐十分开奖结果查询:Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble
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The Mre11-Rad50-Xrs2 (MRX)-Sae2 complex has key functions in initiating the repair of DNA double-strand breaks by homologous recombination. Using purified recombinant proteins, we demonstrate how ATP binding and hydrolysis by the Rad50 subunit regulate the nuclease activities of Mre11. We reconstitute a reaction wherein the MRX-Sae2 ensemble preferentially degrades the 5′-terminated DNA strands at DNA break sites up to several hundred nucleotides in length. This polarity of DNA end processing is required for homologous recombination. Our experiments help explain how MRX-Sae2 and their homologs contribute to the maintenance of genome stability.
To repair DNA double-strand breaks by homologous recombination, the 5′-terminated DNA strands must first be resected to produce 3′ overhangs. Mre11 from Saccharomyces cerevisiae is a 3′ → 5′ exonuclease that is responsible for 5′ end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5′-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3′ end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3′ → 5′ exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5′-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3′ → 5′ degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5′-terminated DNA.
?1E.C. and G.R. contributed equally to this work.
- ?2To whom correspondence should be addressed. Email: .
Author contributions: P.C. designed research; E.C. and G.R. performed research; E.C., G.R., and P.C. analyzed data; and E.C., G.R., and P.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1820157116/-/DCSupplemental.
Published under the PNAS license.