Abstract
Although histone modifications have been implicated in many DNA-dependent processes, their precise role in DNA replication remains largely unknown. Here we describe an efficient single-step method to specifically purify histones located around an origin of replication from Saccharomyces cerevisiae. Using high-resolution MS, we have obtained a comprehensive view of the histone modifications surrounding the origin of replication throughout the cell cycle. We have discovered that acetylation of histone H3 and H4 is dynamically regulated around an origin of replication, at the level of multiply acetylated histones. Furthermore, we find that this acetylation is required for efficient origin activation during S phase.
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Acknowledgements
We are grateful to S. Biggins, B. Akiyoshi and N. Ebel for technical help and advice in developing the TALO8 purification scheme, L.N. Jones and J. Hogan of the Proteomics Facility at the Fred Hutchinson Cancer Research Center for help with MS, J. Eng and B. MacLean for advice with using X!Tandem, T. Kwong (Fred Hutchinson Cancer Research Center), D. Koshland (Carnegie Institute), B. Brewer and M.K. Raghuraman (Univ. of Washington) for the plasmids used in the plasmid-loss assay, and members of the Tsukiyama laboratory S. Parkhurst, S. Biggins, B. Akiyoshi and C. Nelson for helpful comments on the manuscript. This work is supported in part by US National Institutes of Health grant R01 GM078259 to T.T. The Fred Hutchinson Cancer Research Center Proteomics Facility is supported by Cancer Center support grant P30 CA15704.
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A.U. performed the experiments, with help from P.R.G. on MS analyses; T.T. supervised the experiments; A.U. and T.T. wrote the manuscript; all authors discussed the results and approved the manuscript.
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Unnikrishnan, A., Gafken, P. & Tsukiyama, T. Dynamic changes in histone acetylation regulate origins of DNA replication. Nat Struct Mol Biol 17, 430–437 (2010). https://doi.org/10.1038/nsmb.1780
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DOI: https://doi.org/10.1038/nsmb.1780
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