Stepwise 5' DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

Cannavo, Elda; Reginato, Giordano; Cejka, Petr

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Journal article

Stepwise 5' DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

Cannavo, Elda Institute for Research in Biomedicine (IRB), Faculty of Biomedical Sciences, Università della Svizzera italiana, Switzerland
Reginato, Giordano Institute for Research in Biomedicine (IRB), Faculty of Biomedical Sciences, Università della Svizzera italiana, Switzerland - Department of Biology, Institute of Biochemistry, ETH Zurich, Switzerland
Cejka, Petr Institute for Research in Biomedicine (IRB), Faculty of Biomedical Sciences, Università della Svizzera italiana, Switzerland - Department of Biology, Institute of Biochemistry, ETH Zurich, Switzerland

28.02.2019

Published in:

Proceedings of the national academy of sciences of the United States of America. - 2019, vol. 116, no. 12 (March 19), p. 5505-5513

English 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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