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Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae

Nature volume 438, pages 379–383 (2005)Cite this article

Abstract

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. Eukaryotic cells repair DSBs by both non-homologous end joining and homologous recombination. How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important issues. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after DSB formation1, spreads over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci2. In Saccharomyces cerevisiae, phosphorylation of the two principal H2A species is also signalled by DSB formation, which spreads ∼40 kb in either direction from the DSB3. Here we show that near a DSB phosphorylation of H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease; however, phosphorylation of H2A and nucleosome loss occur independently. The DNA damage sensor MRX4 is required for histone loss, which also depends on INO80, a nucleosome remodelling complex5. The repair protein Rad51 (ref. 6) shows delayed recruitment to DSBs in the absence of histone loss, suggesting that MRX-dependent nucleosome remodelling regulates the accessibility of factors directly involved in DNA repair by homologous recombination. Thus, MRX may regulate two pathways of chromatin changes: nucleosome displacement for efficient recruitment of homologous recombination proteins; and phosphorylation of H2A, which modulates checkpoint responses to DNA damage2.

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Figure 1: Chromatin changes at the MATα DSB.
Figure 2: MRX is involved in histone loss at the MATα DSB.
Figure 3: The INO80 complex is required for histone eviction at the MATα DSB.
Figure 4: MRX and INO80 are required for recruiting Rad51 to the MATα DSB.

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Acknowledgements

We thank J. Haber, X. Shen, J. Downs, M. Christman and V. Zakian for strains or plasmids; W.-D. Heyer for antibodies against RPA and Rad51; C. Hillyer and N. Clark for technical assistance; and C.-F. Kao for comments. This work was supported by grants from the NIH. (to M.A.O. and to J.A.N.).

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Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, 915 Camino de Salud, New Mexico, 87131, Albuquerque, USA

    Toyoko Tsukuda, Alastair B. Fleming, Jac A. Nickoloff & Mary Ann Osley

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  3. Jac A. Nickoloff
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Correspondence to Mary Ann Osley.

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Supplementary information

Supplementary Methods

Additional details of the methods used in this study (DOC 33 kb)

Supplementary Figure Legends

Legends to accompany the below Supplementary Figures. (DOC 29 kb)

Supplementary Figure 1

Densitometry traces of MNase ladders at MATα. (PDF 61 kb)

Supplementary Figure 2

MNase digestion of bulk chromatin. (PDF 554 kb)

Supplementary Figure 3

γ-H2A levels and histone eviction profiles in mre11δ and arp8δ mutants. (PDF 107 kb)

Supplementary Figure 4a

MATα DNA is cleaved and resected in an arp8δ mutant. (PDF 5895 kb)

Supplementary Figure 4b,c

MATα DNA is cleaved and resected in an arp8δ mutant. (PDF 57 kb)

Supplementary Figure 5

Role of γ-H2A in recruitment of Ino80 to the MATα DSB. (PDF 68 kb)

Supplementary Figure 6

DNA repair phenotypes of an arp8δ mutant. (PDF 130 kb)

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Tsukuda, T., Fleming, A., Nickoloff, J. et al. Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae. Nature 438, 379–383 (2005). https://doi.org/10.1038/nature04148

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