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A global profile of replicative polymerase usage

Abstract

Three eukaryotic DNA polymerases are essential for genome replication. Polymerase (Pol) α–primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polɛ replicates the leading strand, whereas Polδ performs lagging-strand synthesis. However, it is not known whether this division of labor is maintained across the whole genome or how uniform it is within single replicons. Using Schizosaccharomyces pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome wide. Pu-seq provides direct replication-origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labor is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose that this results from occasional leading-strand initiation by Polδ followed by exchange for Polɛ.

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Figure 1: rNMP incorporation into DNA in Polδ (cdc6-L591G) and Polɛ (cdc20-M630F) cells.
Figure 2: Polymerase usage across the fission-yeast genome.
Figure 3: Identification of replication origins.
Figure 4: Genome replication timing in S. pombe.
Figure 5: Characterization of DNA-replication profiles.
Figure 6: Asymmetric polymerase usage within a replicon.

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Acknowledgements

We thank J. Murray and S. Mohebi for assistance with elutriation and T. Kunkel for advice on polymerase mutation. A.M.C. acknowledges UK Medical Research Council grant G1100074 and European Research Council grant 268788-SMI-DDR. C.A.N. acknowledges Biotechnology and Biological Sciences Research Council grant BB/K007211/1. Y.D. acknowledges a postdoctoral fellowship for research abroad from the Japan Society for the Promotion of Science.

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Authors and Affiliations

Authors

Contributions

A.M.C. conceived the study. A.M.C., I.M., Y.D., C.A.M., A.K., M.H. and C.A.N. designed the experimental and computational approaches. Y.D., I.M., C.A.N., C.A.M., A.K., T.B. and R.R. performed experiments and analysis. A.M.C. wrote the manuscript. C.A.N., Y.D. and A.K. edited the manuscript.

Corresponding authors

Correspondence to Conrad A Nieduszynski or Antony M Carr.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Distribution of replication-origin and termination efficiencies.

(a) The distribution of origin efficiencies (Efori). (b) The distribution of termination frequencies in each 1 kb bin. (c) Left: the observed Polδ bias is maintained in a rad18Δ (rhp18Δ) mutant in which PCNA cannot be modified. Right: replication timing of the three chromosomes (see Fig. 5a). The regions considered “early” replicating are shown in pink and correspond to the pink regions in the vertical bar on the heat map.

Supplementary Figure 2 A genome-wide plot of the polymerase usage.

The top two panels show Polɛ (cdc20-M630F; red), and Polδ (cdc6-L519G; blue) usage on the Watson and Crick strands. The third panel shows the BrdU sequence mapping of origins and red bars indicating the position and efficiency (height) of origins identified from the polymerase usage data. The bottom panel shows median replication timing (Trep) derived from the synchronous culture marker frequency analysis (Fig. 4a).

Supplementary Figure 3 Sequence composition of ribonucleotide incorporation.

Analysis of the sequence composition of ribonucleotide incorporation and the +3 and -3 flanking nucleotides for the wild type polymerase, cdc6-L591G (Polδ) and cdc20-M630F (Polɛ) mutants in the rnh201Δ background.

Supplementary Figure 4 Analysis of nucleotide incorporation into gene coding regions.

(a) Ribonucleotide incorporation into the open reading frame (CDS), the 5’ and 3’ untranslated regions (UTR) and 500bp upstream of the transcription start site (promoter) according to the annotations from the Broad Institute. Data divided by gene size (b) (longer or shorter than 3000 bp, long and short, respectively) and transcript level (c) All transcriptional units are categorised into high or low level of transcription groups derived from table S4 of Marguerat, S. et al. Cell 151, 671–683, 2012, with a threshold of 125. The wide spread of the data corresponding to the high transcript level group is due to the relatively low number (149) of highly transcribed units.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 2 and 3 (PDF 2373 kb)

Supplementary Table 1

Origin efficiencies and location (XLS 186 kb)

Supplementary Data Set 1

Southern blots used in Fig. 1b (PDF 63 kb)

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Daigaku, Y., Keszthelyi, A., Müller, C. et al. A global profile of replicative polymerase usage. Nat Struct Mol Biol 22, 192–198 (2015). https://doi.org/10.1038/nsmb.2962

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