Abstract
The repair of DNA double-strand breaks (DSBs) by homologous recombination is essential for genomic stability. The first step in this process is resection of 5′ strands to generate 3′ single-stranded DNA intermediates. Efficient resection in budding yeast requires the Mre11–Rad50–Xrs2 (MRX) complex and the Sae2 protein, although the role of MRX has been unclear because Mre11 paradoxically has 3′→5′ exonuclease activity in vitro. Here we reconstitute resection with purified MRX, Sae2 and Exo1 proteins and show that degradation of the 5′ strand is catalyzed by Exo1 yet completely dependent on MRX and Sae2 when Exo1 levels are limiting. This stimulation is mainly caused by cooperative binding of DNA substrates by Exo1, MRX and Sae2. This work establishes the direct role of MRX and Sae2 in promoting the resection of 5′ strands in DNA DSB repair.
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Acknowledgements
We are grateful to M. Liskay (Oregon Health and Science University) for yeast Exo1 reagents, to S. Brill (Rutgers University) for RPA antibodies and M. Wold (University of Iowa) for RPA constructs, and to members of the Paull lab for critical comments about this project. This work was supported by US National Institutes of Health grant R01 CA094008 to T.T.P. Studies carried out in the Lee laboratory were supported by R01 GM083010, and S.E.L. is a scholar of the Leukemia and Lymphoma Society.
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M.L.N. expressed and purified recombinant proteins, performed resection experiments in vitro, analyzed the data and helped to edit the manuscript. K.L. performed resection experiments in vivo in S. cerevisiae. Z.G. carried out quantitative PCR analysis of resection products in vitro and contributed to the editing of the manuscript. M.R. performed the SPR experiment and analyzed the SPR data. J.M.C. expressed and purified Sae2 proteins used in this study. S.E.L. helped to design the in vivo experiments with sae2 mutants and helped in the analysis of the data and editing of the manuscript. T.T.P. performed the DNA binding and some of the resection experiments in vitro, analyzed the data, and wrote and edited the manuscript.
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Nicolette, M., Lee, K., Guo, Z. et al. Mre11–Rad50–Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks. Nat Struct Mol Biol 17, 1478–1485 (2010). https://doi.org/10.1038/nsmb.1957
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DOI: https://doi.org/10.1038/nsmb.1957
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