Article | Published:

A global profile of replicative polymerase usage

Nature Structural & Molecular Biology volume 22, pages 192198 (2015) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

References

  1. 1.

    et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864–870 (2005).

  2. 2.

    et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434, 907–913 (2005).

  3. 3.

    & DNA replication timing. Cold Spring Harb. Perspect. Biol. 5, a010132 (2013).

  4. 4.

    Origin recognition and the chromosome cycle. FEBS Lett. 579, 877–884 (2005).

  5. 5.

    et al. High-resolution replication profiles define the stochastic nature of genome replication initiation and termination. Cell Reports 5, 1132–1141 (2013).

  6. 6.

    et al. Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types. Genome Res. 20, 761–770 (2010).

  7. 7.

    , & Principles and concepts of DNA replication in bacteria, archaea, and eukarya. Cold Spring Harb. Perspect. Biol. 5, a010108 (2013).

  8. 8.

    , & Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc. Natl. Acad. Sci. USA 103, 10236–10241 (2006).

  9. 9.

    et al. A key role for Ctf4 in coupling the MCM2–7 helicase to DNA polymerase alpha within the eukaryotic replisome. EMBO J. 28, 2992–3004 (2009).

  10. 10.

    et al. A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome. Nature 510, 293–297 (2014).

  11. 11.

    , , & Dpb2 integrates the leading-strand DNA polymerase into the eukaryotic replisome. Curr. Biol. 23, 543–552 (2013).

  12. 12.

    , , , & DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast. Mol. Biol. Cell 23, 3240–3253 (2012).

  13. 13.

    , , , & CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol ɛ, and GINS in budding yeast. Genes Dev. 24, 602–612 (2010).

  14. 14.

    , , , & Yeast DNA polymerase epsilon participates in leading-strand DNA replication. Science 317, 127–130 (2007).

  15. 15.

    , , , & Division of labor at the eukaryotic replication fork. Mol. Cell 30, 137–144 (2008).

  16. 16.

    , & The major roles of DNA polymerases epsilon and delta at the eukaryotic replication fork are evolutionarily conserved. PLoS Genet. 7, e1002407 (2011).

  17. 17.

    et al. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc. Natl. Acad. Sci. USA 107, 4949–4954 (2010).

  18. 18.

    et al. RNase H2-initiated ribonucleotide excision repair. Mol. Cell 47, 980–986 (2012).

  19. 19.

    et al. Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase I. Science 332, 1561–1564 (2011).

  20. 20.

    et al. Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA. Mol. Cell 49, 1010–1015 (2013).

  21. 21.

    & Site-specific ribonuclease activity of eukaryotic DNA topoisomerase I. Mol. Cell 1, 89–97 (1997).

  22. 22.

    , , & Replication of ribonucleotide-containing DNA templates by yeast replicative polymerases. DNA Repair (Amst.) 10, 897–902 (2011).

  23. 23.

    & Ribonucleotides in DNA: origins, repair and consequences. DNA Repair (Amst.) 19, 27–37 (2014).

  24. 24.

    , , , & Ribonucleotides are signals for mismatch repair of leading-strand replication errors. Mol. Cell 50, 437–443 (2013).

  25. 25.

    et al. Genome-wide identification and characterization of replication origins by deep sequencing. Genome Biol. 13, R27 (2012).

  26. 26.

    , & Heat stress activates fission yeast Spc1/StyI MAPK by a MEKK-independent mechanism. Mol. Biol. Cell 9, 1339–1349 (1998).

  27. 27.

    et al. The dynamics of genome replication using deep sequencing. Nucleic Acids Res. 42, e3 (2014).

  28. 28.

    , & Mathematical modeling of genome replication. Phys. Rev. E 86, 031916 (2012).

  29. 29.

    et al. Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation. Nat. Struct. Mol. Biol. (26 January 2015).

  30. 30.

    , , & Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA. Nat. Methods (26 January 2015).

  31. 31.

    et al. Lagging strand replication shapes the mutational landscape of the genome. Nature (26 January 2015).

  32. 32.

    , , & OriDB, the DNA replication origin database updated and extended. Nucleic Acids Res. 40, D682–D686 (2012).

  33. 33.

    et al. Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements. Mol. Cell 39, 595–605 (2010).

  34. 34.

    & Mutation rates across budding yeast chromosome VI are correlated with replication timing. Genome Biol. Evol. 3, 799–811 (2011).

  35. 35.

    & Readers of PCNA modifications. Chromosoma 122, 259–274 (2013).

  36. 36.

    , & Analysis of the essential functions of the C-terminal protein/protein interaction domain of Saccharomyces cerevisiae pol epsilon and its unexpected ability to support growth in the absence of the DNA polymerase domain. J. Biol. Chem. 274, 22283–22288 (1999).

  37. 37.

    & Schizosaccharomyces pombe cells lacking the amino-terminal catalytic domains of DNA polymerase epsilon are viable but require the DNA damage checkpoint control. Mol. Cell. Biol. 21, 4495–4504 (2001).

  38. 38.

    et al. Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork. Nat. Struct. Mol. Biol. 21, 664–670 (2014).

  39. 39.

    , & Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).

  40. 40.

    , , , & Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe. Gene 407, 63–74 (2008).

  41. 41.

    , & The mechanism of the alkaline hydrolysis of ribonucleic acids. J. Am. Chem. Soc. 76, 2871–2872 (1954).

  42. 42.

    , , & Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection. Silence 3, 9 (2012).

Download references

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.

Author information

Author notes

    • Yasukazu Daigaku
    •  & Andrea Keszthelyi

    These authors contributed equally to this work.

Affiliations

  1. Genome Damage and Stability Centre, University of Sussex, Brighton, UK.

    • Yasukazu Daigaku
    • , Andrea Keszthelyi
    • , Izumi Miyabe
    •  & Antony M Carr
  2. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

    • Carolin A Müller
    •  & Conrad A Nieduszynski
  3. Centre for Translational Omics, University College London Institute of Child Health, London, UK.

    • Tony Brooks
    •  & Mike Hubank
  4. School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham, UK.

    • Renata Retkute

Authors

  1. Search for Yasukazu Daigaku in:

  2. Search for Andrea Keszthelyi in:

  3. Search for Carolin A Müller in:

  4. Search for Izumi Miyabe in:

  5. Search for Tony Brooks in:

  6. Search for Renata Retkute in:

  7. Search for Mike Hubank in:

  8. Search for Conrad A Nieduszynski in:

  9. Search for Antony M Carr in:

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Conrad A Nieduszynski or Antony M Carr.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Tables 2 and 3

  2. 2.

    Supplementary Data Set 1

    Southern blots used in Fig. 1b

Excel files

  1. 1.

    Supplementary Table 1

    Origin efficiencies and location

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.2962

Further reading

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