Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 116, Issue 12, Pages 5505-5513Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1820157116
Keywords
DNA; nuclease; homologous recombination; Mre11; DNA end resection
Categories
Funding
- Swiss National Science Foundation [31003A_175444]
- European Research Council [681630]
- Swiss National Science Foundation (SNF) [31003A_175444] Funding Source: Swiss National Science Foundation (SNF)
- European Research Council (ERC) [681630] Funding Source: European Research Council (ERC)
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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.
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