Abstract
Chromosomal double-strand breaks (DSBs) are resected by 5′ nucleases to form 3′ single-stranded DNA substrates for binding by homologous recombination and DNA damage checkpoint proteins. Two redundant pathways of extensive resection have been described both in cells1,2,3 and in vitro4,5,6, one relying on Exo1 exonuclease and the other on Sgs1 helicase and Dna2 nuclease. However, it remains unknown how resection proceeds within the context of chromatin, where histones and histone-bound proteins represent barriers for resection enzymes. Here we identify the yeast nucleosome-remodelling enzyme Fun30 as a factor promoting DSB end resection. Fun30 is the major nucleosome remodeller promoting extensive Exo1- and Sgs1-dependent resection of DSBs. The RSC and INO80 chromatin-remodelling complexes and Fun30 have redundant roles in resection adjacent to DSB ends. ATPase and helicase domains of Fun30, which are needed for nucleosome remodelling7, are also required for resection. Fun30 is robustly recruited to DNA breaks and spreads along the DSB coincident with resection. Fun30 becomes less important for resection in the absence of the histone-bound Rad9 checkpoint adaptor protein known to block 5′ strand processing8 and in the absence of either histone H3 K79 methylation or γ-H2A, which mediate recruitment of Rad9 (refs 9, 10). Together these data suggest that Fun30 helps to overcome the inhibitory effect of Rad9 on DNA resection.
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Acknowledgements
We thank P. Sung, W.-D. Heyer and A. Pellicioli for antibodies; S.-E. Lee, S. Kron and M. Osley for strains; B. Llorente for sharing unpublished data; and J. Tyler, P. Sung and S. E. Lee for comments on the manuscript. This work was supported by the National Institutes of Health grants GM080600 (to G.I.) and HG004840 (to X.P.).
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X.C. and D.C. contributed equally to this work. X.C. constructed most of the strains and analysed chromatin structure, protein interactions, protein recruitment to DSBs, histone loss at DSBs, activation of the DNA damage checkpoint and DNA damage sensitivity. X.Z. and K.C. performed microarray analysis of gene expression. D.C., J.T. and X.P. performed the genetic screen and constructed FUN30 point mutants. A.P. and C.-D.C. analysed resection and crossover frequency. X.C., X.P. and G.I. designed the experiments, discussed the data and wrote the manuscript.
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Supplementary Information
This file contains Supplementary Tables 2-3, Supplementary References and Supplementary Figures 1-10. (PDF 7524 kb)
Supplementary Data
This file contains Supplementary Table 1 containing all data obtained from the genetic screen. Selected data from this supplemental table are presented in Figure 1 of the main text. (XLS 1268 kb)
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Chen, X., Cui, D., Papusha, A. et al. The Fun30 nucleosome remodeller promotes resection of DNA double-strand break ends. Nature 489, 576–580 (2012). https://doi.org/10.1038/nature11355
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DOI: https://doi.org/10.1038/nature11355
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