Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break


In budding yeast, a single double-strand break (DSB) triggers extensive Tel1 (ATM)- and Mec1 (ATR)-dependent phosphorylation of histone H2A around the DSB, to form γ-H2AX. We describe Mec1- and Tel1-dependent phosphorylation of histone H2B at T129. γ-H2B formation is impaired by γ-H2AX and its binding partner Rad9. High-density microarray analyses show similar γ-H2AX and γ-H2B distributions, but γ-H2B is absent near telomeres. Both γ-H2AX and γ-H2B are strongly diminished over highly transcribed regions. When transcription of GAL7, GAL10 and GAL1 genes is turned off, γ-H2AX is restored within 5 min, in a Mec1-dependent manner; after reinduction of these genes, γ-H2AX is rapidly lost. Moreover, when a DSB is induced near CEN2, γ-H2AX spreads to all other pericentromeric regions, again depending on Mec1. Our data provide new insights in the function and establishment of phosphorylation events occurring on chromatin after DSB induction.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Spreading of γ-H2AX and γ-H2B around an HO-induced DSB.
Figure 2: Profiles of γ-H2AX and γ-H2B in the strain with three HO cuts after DSB induction.
Figure 3: Profiles of γ-H2AX and γ-H2B in undamaged cells.
Figure 4: Differential spreading of γ-H2B in an H2A S129A mutant strain.
Figure 5: Transcriptionally active units are refractory to γ-H2AX and γ-H2B.
Figure 6: γ-H2AX can spread in trans on physically close loci.
Figure 7: Effect of Mec1 and Tel1 kinases on spreading in cis and trans from a DSB induced near CEN2.

Similar content being viewed by others

Accession codes

Primary accessions



  1. Shroff, R. et al. Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr. Biol. 14, 1703–1711 (2004).

    Article  CAS  Google Scholar 

  2. Rogakou, E.P., Boon, C., Redon, C. & Bonner, W.M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916 (1999).

    Article  CAS  Google Scholar 

  3. Harrison, J.C. & Haber, J.E. Surviving the breakup: the DNA damage checkpoint. Annu. Rev. Genet. 40, 209–235 (2006).

    Article  CAS  Google Scholar 

  4. Kim, J.A., Kruhlak, M., Dotiwala, F., Nussenzweig, A. & Haber, J.E. Heterochromatin is refractory to γ-H2AX modification in yeast and mammals. J. Cell Biol. 178, 209–218 (2007).

    Article  CAS  Google Scholar 

  5. Szilard, R.K. et al. Systematic identification of fragile sites via genome-wide location analysis of γ-H2AX. Nat. Struct. Mol. Biol. 17, 299–305 (2010).

    Article  CAS  Google Scholar 

  6. Kitada, T. et al. γH2A is a component of yeast heterochromatin required for telomere elongation. Cell Cycle 10, 293–300 (2011).

    Article  CAS  Google Scholar 

  7. Iacovoni, J.S. et al. High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome. EMBO J. 29, 1446–1457 (2010).

    Article  CAS  Google Scholar 

  8. Caron, P. et al. Cohesin protects genes against γH2AX induced by DNA double-strand breaks. PLoS Genet. 8, e1002460 (2012).

    Article  CAS  Google Scholar 

  9. Melo, J.A., Cohen, J. & Toczyski, D.P. Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. Genes Dev. 15, 2809–2821 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Zou, L. & Elledge, S.J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300, 1542–1548 (2003).

    Article  CAS  Google Scholar 

  11. Nakada, D., Matsumoto, K. & Sugimoto, K. ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism. Genes Dev. 17, 1957–1962 (2003).

    Article  CAS  Google Scholar 

  12. Hammet, A., Magill, C., Heierhorst, J. & Jackson, S.P. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast. EMBO Rep. 8, 851–857 (2007).

    Article  CAS  Google Scholar 

  13. Chen, X. et al. The Fun30 nucleosome remodeler promotes resection of DNA double-strand break ends. Nature 489, 576–580 (2012).

    Article  CAS  Google Scholar 

  14. Eapen, V.V., Sugawara, N., Tsabar, M., Wu, W.H. & Haber, J.E. The Saccharomyces cerevisiae chromatin remodeler Fun30 regulates DNA end resection and checkpoint deactivation. Mol. Cell Biol. 32, 4727–4740 (2012).

    Article  CAS  Google Scholar 

  15. Martin, S.G., Laroche, T., Suka, N., Grunstein, M. & Gasser, S.M. Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast. Cell 97, 621–633 (1999).

    Article  CAS  Google Scholar 

  16. Mills, K.D., Sinclair, D. & Guarente, L. MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks. Cell 97, 609–620 (1999).

    Article  CAS  Google Scholar 

  17. David, L. et al. A high-resolution map of transcription in the yeast genome. Proc. Natl. Acad. Sci. USA 103, 5320–5325 (2006).

    Article  CAS  Google Scholar 

  18. Jamai, A., Imoberdorf, R.M. & Strubin, M. Continuous histone H2B and transcription-dependent histone H3 exchange in yeast cells outside of replication. Mol. Cell 25, 345–355 (2007).

    Article  CAS  Google Scholar 

  19. Moore, J.K. & Haber, J.E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol. Cell Biol. 16, 2164–2173 (1996).

    Article  CAS  Google Scholar 

  20. Ng, H.H., Robert, F., Young, R.A. & Struhl, K. Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol. Cell 11, 709–719 (2003).

    Article  CAS  Google Scholar 

  21. Zhou, B.O. & Zhou, J.Q. Recent transcription-induced histone H3 lysine 4 (H3K4) methylation inhibits gene reactivation. J. Biol. Chem. 286, 34770–34776 (2011).

    Article  CAS  Google Scholar 

  22. Keogh, M.C. et al. A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery. Nature 439, 497–501 (2006).

    Article  CAS  Google Scholar 

  23. Kim, J.A., Hicks, W.M., Li, J., Tay, S.Y. & Haber, J.E. Protein phosphatases pph3, ptc2, and ptc3 play redundant roles in DNA double-strand break repair by homologous recombination. Mol. Cell Biol. 31, 507–516 (2011).

    Article  Google Scholar 

  24. Leroy, C. et al. PP2C phosphatases Ptc2 and Ptc3 are required for DNA checkpoint inactivation after a double-strand break. Mol. Cell 11, 827–835 (2003).

    Article  CAS  Google Scholar 

  25. Morillo-Huesca, M., Clemente-Ruiz, M., Andujar, E. & Prado, F. The SWR1 histone replacement complex causes genetic instability and genome-wide transcription misregulation in the absence of H2A.Z. PLoS ONE 5, e12143 (2010).

    Article  Google Scholar 

  26. Papamichos-Chronakis, M., Krebs, J.E. & Peterson, C.L. Interplay between Ino80 and Swr1 chromatin remodeling enzymes regulates cell cycle checkpoint adaptation in response to DNA damage. Genes Dev. 20, 2437–2449 (2006).

    Article  CAS  Google Scholar 

  27. Halley, J.E., Kaplan, T., Wang, A.Y., Kobor, M.S. & Rine, J. Roles for H2A.Z and its acetylation in GAL1 transcription and gene induction, but not GAL1-transcriptional memory. PLoS Biol. 8, e1000401 (2010).

    Article  Google Scholar 

  28. Lisby, M., Barlow, J.H., Burgess, R.C. & Rothstein, R. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699–713 (2004).

    Article  CAS  Google Scholar 

  29. Ira, G. et al. DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431, 1011–1017 (2004).

    Article  CAS  Google Scholar 

  30. Duan, Z. et al. A three-dimensional model of the yeast genome. Nature 465, 363–367 (2010).

    Article  CAS  Google Scholar 

  31. Renkawitz, J., Lademann, C.A., Kalocsay, M. & Jentsch, S. Monitoring homology search during DNA double-strand break repair in vivo. Mol. Cell 50, 261–272 (2013).

    Article  CAS  Google Scholar 

  32. Myers, J.S. & Cortez, D. Rapid activation of ATR by ionizing radiation requires ATM and Mre11. J. Biol. Chem. 281, 9346–9350 (2006).

    Article  CAS  Google Scholar 

  33. Pellicioli, A., Lee, S.E., Lucca, C., Foiani, M. & Haber, J.E. Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest. Mol. Cell 7, 293–300 (2001).

    Article  CAS  Google Scholar 

  34. Lee, S.E. et al. Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94, 399–409 (1998).

    Article  CAS  Google Scholar 

  35. Bash, R. & Lohr, D. Yeast chromatin structure and regulation of GAL gene expression. Prog. Nucleic Acid Res. Mol. Biol. 65, 197–259 (2001).

    Article  CAS  Google Scholar 

  36. Cavalli, G. & Thoma, F. Chromatin transitions during activation and repression of galactose-regulated genes in yeast. EMBO J. 12, 4603–4613 (1993).

    Article  CAS  Google Scholar 

  37. Radman-Livaja, M. et al. Patterns and mechanisms of ancestral histone protein inheritance in budding yeast. PLoS Biol. 9, e1001075 (2011).

    Article  CAS  Google Scholar 

  38. Li, J. et al. Regulation of budding yeast mating-type switching donor preference by the FHA domain of Fkh1. PLoS Genet. 8, e1002630 (2012).

    Article  CAS  Google Scholar 

  39. Unal, E. et al. DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol. Cell 16, 991–1002 (2004).

    Article  Google Scholar 

  40. Ström, L., Lindroos, H.B., Shirahige, K. & Sjogren, C. Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair. Mol. Cell 16, 1003–1015 (2004).

    Article  Google Scholar 

  41. Bakkenist, C.J. & Kastan, M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003).

    Article  CAS  Google Scholar 

  42. Lee, S.E. et al. Saccharomyces Ku70, Mre11/Rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94, 399–409 (1998).

    Article  CAS  Google Scholar 

  43. Sandell, L.L. & Zakian, V.A. Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75, 729–739 (1993).

    Article  CAS  Google Scholar 

  44. Kim, J.A., Kruhlak, M., Dotiwala, F., Nussenzweig, A. & Haber, J.E. Heterochromatin is refractory to γ-H2AX modification in yeast and mammals. J. Cell Biol. 178, 209–218 (2007).

    Article  CAS  Google Scholar 

  45. Sun, K., Coic, E., Zhou, Z., Durrens, P. & Haber, J.E. Saccharomyces forkhead protein Fkh1 regulates donor preference during mating-type switching through the recombination enhancer. Genes Dev. 16, 2085–2096 (2002).

    Article  CAS  Google Scholar 

  46. Foster, E.R. & Downs, J.A. Methylation of H3 K4 and K79 is not strictly dependent on H2B K123 ubiquitylation. J. Cell Biol. 184, 631–638 (2009).

    Article  CAS  Google Scholar 

  47. Wagschal, A., Delaval, K., Pannetier, M., Arnaud, P. & Feil, R. Chromatin immunoprecipitation (ChIP) on unfixed chromatin from cells and tissues to analyze histone modifications. CSH Protoc. 2007 pdb prot4767 (2007).

Download references


We thanks V. Benes and T. Ivacevic from the European Molecular Biology Laboratory Genomic Core facility for hybridization with Affymetrix arrays. Funding in the Legube laboratory was provided by grants from the Association Contre le Cancer (ARC), Agence Nationale pour la Recherche (ANR-09-JCJC-0138), Canceropole Grand Sud-Ouest and Research Innovation Therapeutic Cancerologie (RITC). Research in the Haber lab was supported by US National Institutes of Health grants GM61766, GM20056 and GM76020.

Author information

Authors and Affiliations



C.-S.L. designed, executed and analyzed experiments shown in Figures 5,6,7 and related supplementary information and prepared ChIP samples for experiments shown in Figures 2,3,4,5,6. K.L. designed, executed and analyzed experiments shown in Figure 1 and related Supplementary Information and prepared ChIP samples for experiments shown in Figures 2,3,4,5,6. G.L. performed ChIP-chip analysis and analyzed the data shown in Figures 2,3,4,5,6 and related supplementary information. J.E.H. designed experiments and analyzed data and was the principal author of the manuscript, with contributions from all other coauthors.

Corresponding authors

Correspondence to Gaëlle Legube or James E Haber.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of antibody to γ-H2B and the efficiency of DSB induction.

(a) A single HO-induced DSB triggered abundant γ-H2AX and γ-H2B modifications in WT cells, shown by western blot. The γ-H2B antibody failed to react to a strain with mutant H2B T129A, while the γ-H2A antibody failed to react to a strain with H2A S129A. (b) Kinetics of HO cleavage at the sites on chromosomes 2, 3 and 6. Cleavage was monitored as a decrease in qPCR signals amplified with primer pairs flanking each cleavage site. The values were normalized to inputs.

Supplementary Figure 2 Profiles of γ-H2AX and γ-H2B.

(a) ChIP-chip were performed with antibodies against γ-H2AX (light blue) and γ-H2B (dark blue), using yeast grown on glucose containing media (no DSB). The profile of γ-H2AX published earlier by Szilard et al, 2010 (purple), is also shown, for comparison. The log2 ratio of ChIP/input signals across four genomic regions are shown. (b) γ-H2AX and γ-H2B are enriched on silent genes in undamaged cells. For each gene of the yeast genome (sacSer1), the averaged γ-H2AX (left panel) or γ-H2B (right panel) signal was calculated on the entire gene length and plotted against the averaged Pol II enrichment (retrieved from David et al, 2006). (c) Differential enrichment of γ-H2AX and γ-H2B at subtelomeric regions. The profiles of γ-H2AX (light blue) and γ-H2B (dark blue) obtained at the left telomere of chromosome 2 (left panel) and on a genomic region farther on the same chromosome (right panel) are shown. Note that, similarly to the signals observed on chromosome 1, shown in Fig. 3a, γ-H2B is less enriched than γ-H2AX at the telomere, while both signals are equivalent further away.

Supplementary Figure 3 Effects of H2A and H2B phosphorylation.

(a) Effect of γ-H2AX and γ-H2B on telomere length. DNA from logarithmically growing cells was purified and digested with XhoI that cleaved within the Y' subtelomeric element. A Southern blot was probed with a Y' probe that hybridized with the terminal (telomere containing) fragment and several sizes of internal subtelomeric Y' repeats. (b) γ-H2B profile is similar in WT and in an H2A S129A mutant strain on undamaged chromosomes. For each gene of the yeast genome (sacSer1), the averaged γ-H2B signal in H2A S129A mutant was calculated on the entire gene length and plotted against the averaged Pol II enrichment (retrieved from David et al, 2006). (c) Examples of the profiles of γ-H2B signal upon glucose growth (no DSB). Both in WT (blue) and in the H2A mutant strain (black). (d) γ-H2AX and γ-H2B profiles near telomeres in cells without and with a single unrepaired DSB. γ-H2AX and γ-H2B ChIP-chip signals in WT or in an H2A S129A mutant strain obtained upon growth on glucose (no DSB) or galactose (HO cut) were averaged on 20 kb at all chromosomes ends (left and right arms combined).

Supplementary Figure 4 γ-H2AX profiles at the GAL gene cluster in response to medium change.

(a) γ-H2AX at several positions within the GAL-encoding gene cluster, as represented in the Saccharomyces Genome Database. Fold increase, calculated by normalizing IP/input value to that at 0 h, for γ-H2AX ChIP are shown for the primer pairs (arrows) at times after galactose induction of a DSB approximately 20 kb to the left of these genes. At 1 h, glucose was added to an aliquot of the culture to repress GAL gene transcription for another one hour. In this experiment, cells were not first washed free of galactose and the levels of increase in γ–H2AX modification are lower than that in Fig. 5. (b) Restoration and displacement of γ-H2AX from the GAL10 gene as a function of repression or re-induction do not depend on the three checkpoint phosphatases, implicated in checkpoint regulation and in dephosphorylating γ-H2AX, nor on histone H2A.Z. (c) H2A level on GAL10 gene did not undergo dramatic changes upon transfer on glucose. Histone H2A levels were determined by ChIP at the GAL10 gene under the conditions described in Fig. 5d. ChIP efficiency was calculated by normalizing IP/input value to that at 0 h. (d) Mec1-dependency on restoration of γ-H2AX at GAL10 after turning off transcription. Cells were arrested with nocodazole before galactose-mediated induction of an unrepairable DSB near the centromere of chromosome 2. γ-H2AX was measured at the GAL10 gene after transcription was turned off at the times indicated by transferring cells to dextrose-containing medium, as described in Figure 5.

Supplementary Figure 5 Distribution of γ-H2AX and γ-H2B around centromeres.

(a) γ-H2AX accumulated at pericentromeric regions of undamaged chromosomes after induction of DSB in a strain with three DSBs. Profile of the γ-H2AX signal obtained upon galactose growth (DSB induction) versus glucose growth (no DSB). All chromosomes except chromosomes 2, 3 and 6 (shown in Fig. 2) and chromosomes 15 and 16 (shown in Fig. 6a) are shown. Centromeres are indicated by an arrow. (b) γ-H2B accumulated at pericentromeric regions of undamaged chromosomes after induction of DSB in a strain with three DSBs. Profile of the γ-H2B signal obtained upon galactose growth (DSB induction) versus glucose growth (no DSB). Centromeres are indicated by an arrow.

Supplementary Figure 6 Effects of H2A and H2B phosphorylation on resection and drug sensitivity.

(a) Effect of phosphorylation of H2A and H2B on 5'-to-3' resection around a DSB. 2 h after HO-induction, the extent of 5'-to-3' resection was measured by chromatin immunoprecipitation of the largest subunit of RPA, Rfa1. The more extensive resection seen in an H2A S129A mutant strain was suppressed by H2B T129A mutatioin. (b) Effect of phosphorylation of H2A and H2B on sensitivity to DNA damaging agents. Serial dilutions were plated on YEPD plates and on agar plates containing various DNA damaging agents or after exposure to UV light. The H2B T129A mutation suppressed both the resistance to phleomycin and the sensitivity to MMS in an H2A S129A mutant strain.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1 and 2 (PDF 3709 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, CS., Lee, K., Legube, G. et al. Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break. Nat Struct Mol Biol 21, 103–109 (2014).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing