Abstract
Post-translational histone modifications are crucial for the regulation of numerous DNA-templated processes, and are thought to mediate both alteration of chromatin dynamics and recruitment of effector proteins to specific regions of the genome1. In particular, histone Ser/Thr phosphorylation regulates multiple nuclear functions in the budding yeast Saccharomyces cerevisiae, including transcription, DNA damage repair, mitosis, apoptosis and sporulation2. Although modifications to chromatin during replication remain poorly understood, a number of recent studies have described acetylation of the histone H3 N-terminal α-helix (αN helix) at Lys 56 as a modification that is important for maintenance of genomic integrity during DNA replication and repair3,4. Here, we report phosphorylation of H3 Thr 45 (H3-T45), a histone modification also located within the H3 αN helix in S. cerevisiae. Thr 45 phosphorylation peaks during DNA replication, and is mediated by the S phase kinase Cdc7–Dbf4 as part of a multiprotein complex identified in this study. Furthermore, loss of phosphorylated H3-T45 causes phenotypes consistent with replicative defects, and prolonged replication stress results in H3-T45 phosphorylation accumulation over time. Notably, the phenotypes described here are independent of Lys 56 acetylation status, and combinatorial mutations to both Thr 45 and Lys 56 of H3 cause synthetic growth defects. Together, these data identify and characterize H3-T45 phosphorylation as a replication-associated histone modification in budding yeast.
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Acknowledgements
We thank members of the Grant and Hunt labs for helpful discussion and technical assistance; R. Sclafani for yeast strains and reagents; G. Kupfer for reagents; J. Smith and J. Reese for yeast strains; and D. Auble for yeast strains, helpful discussion and reading of this manuscript. We also thank J. Bone (Active Motif) for assistance with antibody generation. S.P.B. was supported in part by NIH pre-doctoral cancer training grant no. 5 T32 CA009109-30. This work was supported by grants from the NIH to P.A.G. (5 P30 CA044579-18 and R56 DK082673-01), D.F.H. (GM37537), J.R.Y. (P41 RR011823), and M.M.S. (GM60444).
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S.P.B. performed purifications of TAP-tagged Cdc7 complex, recombinant Cdc7 and Dbf4 expression and purification, in vitro histone and peptide kinase assays, mass spectrometric identification of Thr 45 phosphorylation, antibody characterization, yeast strain generation, mutagenesis, growth assays, synchronization experiments, inductions, and associated western blotting. J.P. identified and isolated the native Cdc7 histone kinase complex, generated the Cdc7–TAP strain and performed the Cdc7 immunoprecipitation and associated kinase assays. S.A. and J.R.Y. performed the mass spectrometric identification of the kinase complex subunits and subsequent data analysis. Q.Q. and M.M.S. performed and analysed the FACS data. J.S. and D.F.H. assisted with the mass spectrometric mapping of the histone phosphorylation mark and data analysis. P.A.G. conducted experimental design and analysis. S.P.B. and P.A.G. wrote the manuscript.
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Baker, S., Phillips, J., Anderson, S. et al. Histone H3 Thr 45 phosphorylation is a replication-associated post-translational modification in S. cerevisiae. Nat Cell Biol 12, 294–298 (2010). https://doi.org/10.1038/ncb2030
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DOI: https://doi.org/10.1038/ncb2030
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