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Intrinsic coupling of lagging-strand synthesis to chromatin assembly

Abstract

Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Our studies represent the first high-resolution analysis—to our knowledge—of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly.

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Figure 1: DNA ligase I depletion in S. cerevisiae leads to the accumulation of Okazaki fragments sized similarly to the nucleosome repeat.
Figure 2: Sequenced Okazaki fragments show a pronounced bias towards the lagging strand.
Figure 3: Okazaki fragment termini are preferentially located at nucleosome dyads.
Figure 4: Transcription factors with roles in nucleosome positioning stimulate dissociation of Pol δ.
Figure 5: Impaired chromatin assembly and Pol δ processivity affect the size of Okazaki fragments and the location of their termini, respectively.

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Primary accessions

Gene Expression Omnibus

Data deposits

Raw sequencing data and processed data are available at theGene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE33786) under accession number 33786.

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Acknowledgements

We thank S. McGuffee for assistance with data processing; S. Keeney, K. Marians, D. Remus, T. Tsukiyama, members of the Molecular Biology Program and Whitehouse laboratory for discussions and comments on the manuscript. This work was supported by a Louis V. Gerstner Jr Young Investigator Award and an Alfred Bressler Scholars Endowment Award to I.W. D.J.S. is an HHMI fellow of the Damon Runyon Cancer Research Foundation (DRG-#2046-10).

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D.J.S. and I.W. designed experiments; D.J.S. performed experiments and analysed data; D.J.S. and I.W. interpreted results; the manuscript was drafted by D.J.S. and edited by D.J.S. and I.W.

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Correspondence to Iestyn Whitehouse.

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Smith, D., Whitehouse, I. Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Nature 483, 434–438 (2012). https://doi.org/10.1038/nature10895

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