Replication fork reactivation downstream of a blocked nascent leading strand

Abstract

Unrepaired lesions in the DNA template pose a threat to accurate replication. Several pathways exist in Escherichia coli to reactivate a blocked replication fork. The process of recombination-dependent restart of broken forks is well understood, but the consequence of replication through strand-specific lesions is less well known. Here we show that replication can be restarted and leading-strand synthesis re-initiated downstream of an unrepaired block to leading-strand progression, even when the 3′-OH of the nascent leading strand is unavailable. We demonstrate that the loading by a replication restart system of a single hexamer of the replication fork helicase, DnaB, on the lagging-strand template is sufficient to coordinate priming by the DnaG primase of both the leading and lagging strands. These observations provide a mechanism for damage bypass during fork reactivation, demonstrate how daughter-strand gaps are generated opposite leading-strand lesions during the replication of ultraviolet-light-irradiated DNA, and help to explain the remarkable speed at which even a heavily damaged DNA template is replicated.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The leading strand can be reprimed during both PriA- and PriC-dependent restart.
Figure 2: A modified linear template in which the fork 3′-arm is replaced with a biotin group.
Figure 3: A single DnaB hexamer on the lagging-strand template coordinates priming of both strands.
Figure 4: PriC-dependent restart of a stalled fork generates daughter-strand gaps.

References

  1. 1

    Myung, K., Chen, C. & Kolodner, R. D. Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae. Nature 411, 1073–1076 (2001)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Witkin, E. M. Ultraviolet-induced mutation and DNA repair. Annu. Rev. Microbiol. 23, 487–514 (1969)

    CAS  Article  Google Scholar 

  3. 3

    Rupp, W. D. & Howard-Flanders, P. Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J. Mol. Biol. 31, 291–304 (1968)

    CAS  Article  Google Scholar 

  4. 4

    Iyer, V. N. & Rupp, W. D. Usefulness of benzoylated naphthoylated DEAE-cellulose to distinguish and fractionate double-stranded DNA bearing different extents of single-stranded regions. Biochim. Biophys. Acta 228, 117–126 (1971)

    CAS  Article  Google Scholar 

  5. 5

    Bridges, B. A. & Sedgwick, S. G. Effect of photoreactivation on the filling of gaps in deoxyribonucleic acid synthesized after exposure of Escherichia coli to ultraviolet light. J. Bacteriol. 117, 1077–1081 (1974)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Ganesan, A. K. Persistence of pyrimidine dimers during post-replication repair in ultraviolet light-irradiated Escherichia coli K12. J. Mol. Biol. 87, 103–119 (1974)

    CAS  Article  Google Scholar 

  7. 7

    Bridges, B. A. & Munson, R. J. Mutagenesis in Escherichia coli: evidence for the mechanism of base change mutation by ultraviolet radiation in a strain deficient in excision-repair. Proc. R. Soc. Lond. B 171, 213–226 (1968)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Higuchi, K. et al. Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro. Genes Cells 8, 437–449 (2003)

    CAS  Article  Google Scholar 

  9. 9

    McInerney, P. & O'Donnell, M. Functional uncoupling of twin polymerases: mechanism of polymerase dissociation from a lagging-strand block. J. Biol. Chem. 279, 21543–21551 (2004)

    CAS  Article  Google Scholar 

  10. 10

    Pages, V. & Fuchs, R. P. Uncoupling of leading- and lagging-strand DNA replication during lesion bypass in vivo. Science 300, 1300–1303 (2003)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Higgins, N. P., Kato, K. & Strauss, B. A model for replication repair in mammalian cells. J. Mol. Biol. 101, 417–425 (1976)

    CAS  Article  Google Scholar 

  12. 12

    Seigneur, M., Bidnenko, V., Ehrlich, S. D. & Michel, B. RuvAB acts at arrested replication forks. Cell 95, 419–430 (1998)

    CAS  Article  Google Scholar 

  13. 13

    Sandler, S. J. & Marians, K. J. Role of PriA in replication fork reactivation in Escherichia coli. J. Bacteriol. 182, 9–13 (2000)

    CAS  Article  Google Scholar 

  14. 14

    Michel, B., Grompone, G., Flores, M. J. & Bidnenko, V. Multiple pathways process stalled replication forks. Proc. Natl Acad. Sci. USA 101, 12783–12788 (2004)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Heller, R. C. & Marians, K. J. The disposition of nascent strands at stalled replication forks dictates the pathway of replisome loading during restart. Mol. Cell 17, 733–743 (2005)

    CAS  Article  Google Scholar 

  16. 16

    Wu, C. A., Zechner, E. L. & Marians, K. J. Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size. J. Biol. Chem. 267, 4030–4044 (1992)

    CAS  PubMed  Google Scholar 

  17. 17

    Xu, L. & Marians, K. J. PriA mediates DNA replication pathway choice at recombination intermediates. Mol. Cell 11, 817–826 (2003)

    CAS  Article  Google Scholar 

  18. 18

    Swart, J. R. & Griep, M. A. Primase from Escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins. Template sequence requirements based on the bacteriophage G4 complementary strand origin and Okazaki fragment initiation sites. J. Biol. Chem. 268, 12970–12976 (1993)

    CAS  PubMed  Google Scholar 

  19. 19

    Tougu, K., Peng, H. & Marians, K. J. Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork. J. Biol. Chem. 269, 4675–4682 (1994)

    CAS  PubMed  Google Scholar 

  20. 20

    Hacker, K. J. & Johnson, K. A. A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding. Biochemistry 36, 14080–14087 (1997)

    CAS  Article  Google Scholar 

  21. 21

    Kaplan, D. L. & O'Donnell, M. DnaB drives DNA branch migration and dislodges proteins while encircling two DNA strands. Mol. Cell 10, 647–657 (2002)

    CAS  Article  Google Scholar 

  22. 22

    Xu, L. & Marians, K. J. Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function. J. Biol. Chem. 275, 8196–8205 (2000)

    CAS  Article  Google Scholar 

  23. 23

    Galletto, R., Jezewska, M. J. & Bujalowski, W. Interactions of the Escherichia coli DnaB helicase hexamer with the replication factor the DnaC protein. Effect of nucleotide cofactors and the ssDNA on protein-protein interactions and the topology of the complex. J. Mol. Biol. 329, 441–465 (2003)

    CAS  Article  Google Scholar 

  24. 24

    Davey, M. J., Fang, L., McInerney, P., Georgescu, R. E. & O'Donnell, M. The DnaC helicase loader is a dual ATP/ADP switch protein. EMBO J. 21, 3148–3159 (2002)

    CAS  Article  Google Scholar 

  25. 25

    Reha-Krantz, L. J. & Hurwitz, J. The dnaB gene product of Escherichia coli. II. Single stranded DNA-dependent ribonucleoside triphosphatase activity. J. Biol. Chem. 253, 4051–4057 (1978)

    CAS  PubMed  Google Scholar 

  26. 26

    Mitkova, A. V., Khopde, S. M. & Biswas, S. B. Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis. J. Biol. Chem. 278, 52253–52261 (2003)

    CAS  Article  Google Scholar 

  27. 27

    Smith, K. C. Recombinational DNA repair: the ignored repair systems. Bioessays 26, 1322–1326 (2004)

    CAS  Article  Google Scholar 

  28. 28

    Grompone, G., Sanchez, N., Dusko Ehrlich, S. & Michel, B. Requirement for RecFOR-mediated recombination in priA mutant. Mol. Microbiol. 52, 551–562 (2004)

    CAS  Article  Google Scholar 

  29. 29

    Sandler, S. J. Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12. Genetics 155, 487–497 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Prakash, L. Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations. Mol. Gen. Genet. 184, 471–478 (1981)

    CAS  Article  Google Scholar 

  31. 31

    Lehmann, A. R. Postreplication repair of DNA in ultraviolet-irradiated mammalian cells. J. Mol. Biol. 66, 319–337 (1972)

    CAS  Article  Google Scholar 

  32. 32

    Meneghini, R. Gaps in DNA synthesized by ultraviolet light-irradiated WI38 human cells. Biochim. Biophys. Acta 425, 419–427 (1976)

    CAS  Article  Google Scholar 

  33. 33

    Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K. & Sugino, A. Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc. Natl Acad. Sci. USA 59, 598–605 (1968)

    ADS  CAS  Article  Google Scholar 

  34. 34

    Ogawa, T. & Okazaki, T. Discontinuous DNA replication. Annu. Rev. Biochem. 49, 421–457 (1980)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

These studies were supported by a grant from the NIH.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Kenneth J. Marians.

Ethics declarations

Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Heller, R., Marians, K. Replication fork reactivation downstream of a blocked nascent leading strand. Nature 439, 557–562 (2006). https://doi.org/10.1038/nature04329

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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