Skip to main content
SpringerLink
Log in

DNA Replication Fork Proteins

  • Protocol
  • First Online:
DNA Replication

Part of the book series: Methods in Molecular Biology ((MIMB,volume 521))

Summary

DNA replication is a complex mechanism that functions due to the co-ordinated interplay of several dozen protein factors. In the last few years, numerous studies suggested a tight implication of DNA replication factors in several DNA transaction events that maintain the integrity of the genome. Therefore, DNA replication fork proteins have also to be considered as part of a general process aiming at replicating and protecting the genome in order to allow the correct function of a cell and of its eventual daughter cells. This is illustrated by several DNA repair pathways such as base excision repair, nucleotide excision repair, double-strand break repair, and mismatch repair. Furthermore, several of the replication proteins have also been shown to be essential in sensing and transducing DNA damages through the checkpoint cascade pathways. This review will summarize the properties of DNA replication proteins that function exclusively at the replication fork.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Garg, P., and Burgers, P. M. (2005) DNA polymerases that propagate the eukaryotic DNA replication fork, Crit Rev Biochem Mol Biol 40, 115–128.

    Article  PubMed  CAS  Google Scholar 

  2. Hubscher, U., Maga, G., and Spadari, S. (2002) Eukaryotic DNA polymerases, Annu Rev Biochem 71, 133–163.

    Article  PubMed  CAS  Google Scholar 

  3. Johnson, A., and O’Donnell, M. (2005) Cellular DNA replicases: components and dynamics at the replication fork, Annu Rev Biochem 74, 283–315.

    Article  PubMed  CAS  Google Scholar 

  4. Maga, G., Villani, G., Tillement, V., Stucki, M., Locatelli, G. A., Frouin, I., Spadari, S., and Hübscher, U. (2001) Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase δ by the concerted action of replication protein A, proliferating cell nuclear antigen and flap endonuclease 1., Proc Natl Acad Sci USA 98, 14298–14303.

    Article  PubMed  CAS  Google Scholar 

  5. Waga, S., and Stillman, B. (1998) The DNA replication fork in eukaryotic cells, Annu Rev Biochem 67, 721–751.

    Article  PubMed  CAS  Google Scholar 

  6. Kearsey, S. E., and Cotterill, S. (2003) Enigmatic variations: divergent modes of regulating eukaryotic DNA replication, Mol Cell 12, 1067–1075.

    Article  PubMed  CAS  Google Scholar 

  7. Machida, Y. J., and Dutta, A. (2007) The APC/C inhibitor, Emi1, is essential for prevention of rereplication, Genes Dev 21, 184–194.

    Article  PubMed  CAS  Google Scholar 

  8. Zhu, W., Ukomadu, C., Jha, S., Senga, T., Dhar, S. K., Wohlschlegel, J. A., Nutt, L. K., Kornbluth, S., and Dutta, A. (2007) Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication, Genes Dev 21, 2288–2299.

    Article  PubMed  CAS  Google Scholar 

  9. Shevelev, I. V., and Hubscher, U. (2002) The 3′→5′ exonucleases, Nat Rev Mol Cell Biol 3, 364–376.

    Article  PubMed  CAS  Google Scholar 

  10. Wang, J. C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective, Nat Rev Mol Cell Biol 3, 430–440.

    Article  PubMed  CAS  Google Scholar 

  11. Labib, K., and Gambus, A. (2007) A key role for the GINS complex at DNA replication forks, Trends Cell Biol 17, 271–278.

    Article  PubMed  CAS  Google Scholar 

  12. Zegerman, P., and Diffley, J. F. (2007) Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast, Nature 445, 281–285.

    Article  PubMed  CAS  Google Scholar 

  13. DeFalco, S., Ferrari, E., DeFelice, M., Rossi, M., Hübscher, U. and Pisani, F.H. (2007) The human GINS complex binds to and specifically stimulates human DNA polymerase alpha-primase, EMBO Rep 8, 99–103.

    Article  CAS  Google Scholar 

  14. Fanning, E., Klimovich, V., and Nager, A. R. (2006) A dynamic model for replication protein A (RPA) function in DNA processing pathways, Nucleic Acids Res 34, 4126–4137.

    Article  PubMed  CAS  Google Scholar 

  15. Maga, G., Frouin, I., Spadari, S., and Hubscher, U. (2001) Replication protein A as a “fidelity clamp” for DNA polymerase alpha, J Biol Chem 276, 18235–18242.

    Article  PubMed  CAS  Google Scholar 

  16. Yuzhakov, A., Kelman, Z., Hurwitz, J., and O’Donnell, M. (1999) Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme, EMBO J 18, 6189–6199.

    Article  PubMed  CAS  Google Scholar 

  17. Bae, S. H., Bae, K. H., Kim, J. A., and Seo, Y. S. (2001) RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes, Nature 412, 456–461.

    Article  PubMed  CAS  Google Scholar 

  18. Zou, L., Liu, D., and Elledge, S. J. (2003) Replication protein A-mediated recruitment and activation of Rad17 complexes, Proc Natl Acad Sci U S A 100, 13827–13832.

    Article  PubMed  CAS  Google Scholar 

  19. Wang, X., Ira, G., Tercero, J. A., Holmes, A. M., Diffley, J. F., and Haber, J. E. (2004) Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae, Mol Cell Biol 24, 6891–6899.

    Article  PubMed  CAS  Google Scholar 

  20. Maga, G., Stucki, M., Spadari, S., and Hubscher, U. (2000) DNA polymerase switching: I. Replication factor C displaces DNA polymerase alpha prior to PCNA loading, J Mol Biol 295, 791–801.ss

    Article  PubMed  CAS  Google Scholar 

  21. Mossi, R., Keller, R. C., Ferrari, E., and Hubscher, U. (2000) DNA polymerase switching: II. Replication factor C abrogates primer synthesis by DNA polymerase alpha at a critical length, J Mol Biol 295, 803–814.

    Article  PubMed  CAS  Google Scholar 

  22. Bowman, G. D., O’Donnell, M., and Kuriyan, J. (2004) Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex, Nature 429, 724–730.

    Article  PubMed  CAS  Google Scholar 

  23. Miyata, T., Oyama, T. Mayanagi, K., Ishino, S., Ishino, Y., and Morikawa, K. (2004) The clamp-loading complex for processive DNA replication, Nat Struct Mol Biol 11, 632–636.

    Article  PubMed  CAS  Google Scholar 

  24. Jansen, J. G., Fousteri, M. I., and de Wind, N. (2007) Send in the clamps: control of DNA translesion synthesis in eukaryotes, Mol Cell 28, 522–529.

    Article  PubMed  CAS  Google Scholar 

  25. Toueille, M., and Hübscher, U. (2006) PCNA: a landing stage to overcome crisis situations Lee, H. ed. Research Signpost ISBN 81-308-0096-9, pp 83–106.

    Google Scholar 

  26. Moldovan, G. L., Pfander, B., and Jentsch, S. (2007) PCNA, the Maestro of the Replication Fork, Cell 129, 665–679.

    Article  PubMed  CAS  Google Scholar 

  27. Pursell, Z. F., Isoz, I., Lundstrom, E. B., Johansson, E., and Kunkel, T. A. (2007) Yeast DNA polymerase epsilon participates in leading-strand DNA replication, Science 317, 127–130.

    Article  PubMed  CAS  Google Scholar 

  28. Ohya, T., Kawasaki, Y., Hiraga, S., Kanbara, S., Nakajo, K., Nakashima, N., Suzuki, A., and Sugino, A. (2002) The DNA polymerase domain of pol(epsilon) is required for rapid, efficient, and highly accurate chromosomal DNA replication, telomere length maintenance, and normal cell senescence in Saccharomyces cerevisiae, J Biol Chem 277, 28099–28108.

    Article  PubMed  CAS  Google Scholar 

  29. Kesti, T., Flick, K., Keranen, S., Syvaoja, J. E., and Wittenberg, C. (1999) DNA polymerase epsilon catalytic domains are dispensable for DNA replication, DNA repair, and cell viability, Mol Cell 3, 679–685.

    Article  PubMed  CAS  Google Scholar 

  30. Cullmann, G., Hindges, R., Berchtold, M. W., and Hubscher, U. (1993) Cloning of a mouse cDNA encoding DNA polymerase delta: refinement of the homology boxes, Gene 134, 191–200.

    Article  PubMed  CAS  Google Scholar 

  31. Kokoska, R. J., Stefanovic, L., DeMai, J., and Petes, T. D. (2000) Increased rates of genomic deletions generated by mutations in the yeast gene encoding DNA polymerase delta or by decreases in the cellular levels of DNA polymerase delta, Mol Cell Biol 20, 7490–7504.

    Article  PubMed  CAS  Google Scholar 

  32. Goldsby, R. E., Lawrence, N. A., Hays, L. E., Olmsted, E. A., Chen, X., Singh, M., and Preston, B. D. (2001) Defective DNA polymerase-delta proofreading causes cancer susceptibility in mice, Nat Med 7, 638–639.

    Article  PubMed  CAS  Google Scholar 

  33. Goldsby, R. E., Hays, L. E., Chen, X., Olmsted, E. A., Slayton, W. B., Spangrude, G. J., and Preston, B. D. (2002) High incidence of epithelial cancers in mice deficient for DNA polymerase delta proofreading, Proc Natl Acad Sci USA 99, 15560–15565.

    Article  PubMed  CAS  Google Scholar 

  34. Liu, Y., Kao, H. I., and Bambara, R. A. (2004) Flap endonuclease 1: a central component of DNA metabolism, Annu Rev Biochem 73, 589–615.

    Article  PubMed  CAS  Google Scholar 

  35. Henneke, G., Friedrich-Heinecken, E., and Hubscher, U. (2003) Flap endonuclease 1, a novel tumor suppressor protein, Trends Biochem Sci 28, 384–390.

    Article  PubMed  CAS  Google Scholar 

  36. Kucherlapati, M., Yang, K., Kuraguchi, M., Zhao, J., Lia, M., Heyer, J., Kane, M. F., Fan, K., Russell, R., Brown, A. M., Kneitz, B., Edelmann, W., Kolodner, R. D., Lipkin, M., and Kucherlapati, R. (2002) Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression, Proc Natl Acad Sci USA 99, 9924–9929.

    Article  PubMed  CAS  Google Scholar 

  37. Stewart, J. A., Campbell, J. L., and Bambara, R. A. (2006) Flap endonuclease disengages DNA2 helicase/nuclease from Okazaki fragment flaps, J Biol Chem 281, 38565–38572.

    Article  PubMed  CAS  Google Scholar 

  38. Kao, H. I., Campbell, J. L., and Bambara, R. A. (2004) DNA2p helicase/nuclease is a tracking protein, like FEN1, for flap cleavage during Okazaki fragment maturation, J Biol Chem 279, 50840–50849.

    Article  PubMed  CAS  Google Scholar 

  39. Li, X., Li, J., Harrington, J., Lieber, M. R., and Burgers, P. M. (1995) Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen, J Biol Chem 270, 22109–22112.

    Article  PubMed  CAS  Google Scholar 

  40. Jin, Y. H., Ayyagari, R., Resnick, M. A., Gordenin, D. A., and Burgers, P. M. (2003) Okazaki fragment maturation in yeast. II. Cooperation between the polymerase and 3′-5′-exonuclease activities of Pol delta in the creation of a ligatable nick, J Biol Chem 278, 1626–1633.

    Article  PubMed  CAS  Google Scholar 

  41. Hubscher, U., and Seo, Y. S. (2001) Replication of the lagging strand: a concert of at least 23 polypeptides, Mol Cells 12, 149–157.

    PubMed  CAS  Google Scholar 

  42. Jin, Y. H., Garg, P., Stith, C. M., Al-Refai, H., Sterling, J. F., Murray, L. J., Kunkel, T. A., Resnick, M. A., Burgers, P. M., and Gordenin, D. A. (2005) The multiple biological roles of the 3′→5′ exonuclease of Saccharomyces cerevisiae DNA polymerase delta require switching between the polymerase and exonuclease domains, Mol Cell Biol 25, 461–471.

    Article  PubMed  CAS  Google Scholar 

  43. Stucki, M., Jonsson, Z. O., and Hubscher, U. (2001) In eukaryotic flap endonuclease 1, the C terminus is essential for substrate binding, J Biol Chem 276, 7843–7849.

    Article  PubMed  CAS  Google Scholar 

  44. Sakurai, S., Kitano, K., Yamaguchi, H., Hamada, K., Okada, K., Fukuda, K., Uchida, M., Ohtsuka, E., Morioka, H., and Hakoshima, T. (2005) Structural basis for recruitment of human flap endonuclease 1 to PCNA, EMBO J 24, 683–693.

    Article  PubMed  CAS  Google Scholar 

  45. Chapados, B. R., Hosfield, D. J., Han, S., Qiu, J., Yelent, B., Shen, B., and Tainer, J. A. (2004) Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair, Cell 116, 39–50.

    Article  PubMed  CAS  Google Scholar 

  46. Tomkinson, A. E., Vijayakumar, S., Pascal, J. M., and Ellenberger, T. (2006) DNA ligases: structure, reaction mechanism, and function, Chem Rev 106, 687–699.

    Article  PubMed  CAS  Google Scholar 

  47. Levin, D. S., Bai, W., Yao, N., O’Donnell, M., and Tomkinson, A. E. (1997) An interaction between DNA ligase I and proliferating cell nuclear antigen: implications for Okazaki fragment synthesis and joining, Proc Natl Acad Sci USA 94, 12863–12868.

    Article  PubMed  CAS  Google Scholar 

  48. Jonsson, Z. O., Hindges, R., and Hubscher, U. (1998) Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen, Embo J 17, 2412–2425.

    Article  PubMed  CAS  Google Scholar 

  49. Tom, S., Henricksen, L. A., Park, M. S., and Bambara, R. A. (2001) DNA ligase I and proliferating cell nuclear antigen form a functional complex, J Biol Chem 276, 24817–24825.

    Article  PubMed  CAS  Google Scholar 

  50. Song, W., Levin, D. S., Varkey, J., Post, S., Bermudez, V. P., Hurwitz, J., and Tomkinson, A. E. (2007) A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes, J Biol Chem 282, 22721–22730.

    Article  PubMed  CAS  Google Scholar 

  51. Lovett, S. T. (2007) Polymerase switching in DNA replication, Mol Cell 27, 523–526.

    Article  PubMed  CAS  Google Scholar 

  52. Chilkova, O., Stenlund, P., Isoz, I., Stith, C. M., Grabowski, P., Lundstrom, E. B., Burgers, P. M., and Johansson, E. (2007) The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA, Nucleic Acids Res 35, 6588–6597.

    Article  PubMed  CAS  Google Scholar 

  53. McInerney, P., Johnson, A., Katz, F., and O’Donnell, M. (2007) Characterization of a triple DNA polymerase replisome, Mol Cell 27, 527–538.

    Article  PubMed  CAS  Google Scholar 

  54. Langston, L. D., and O’Donnell, M. (2006) DNA replication: keep moving and don’t mind the gap, Mol Cell 23, 155–160.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

I thank Giuseppe Villani for critically reading of the manuscript and Ursula Hübscher for the artwork. Work in the author’s laboratory has been supported by the Swiss National Science Foundation, by the UBS Foundation ‘im Auftrage eines Kunden’ and by the University of Zurich, Switzerland.

Author information

Authors and Affiliations

  1. Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich-Irchel, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland

    Ulrich Hübscher

Authors
  1. Ulrich Hübscher
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ulrich Hübscher .

Editor information

Editors and Affiliations

  1. Marie Curie Research Institute, The Chart, Oxted, RH 8 0TL, United Kingdom

    Sonya Vengrova

  2. Marie Curie Research Institute, The Chart, Oxted, RH 8 0TL, United Kingdom

    Jacob Z. Dalgaard

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Hübscher, U. (2009). DNA Replication Fork Proteins. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 521. Humana Press. https://doi.org/10.1007/978-1-60327-815-7_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-815-7_2

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-814-0

  • Online ISBN: 978-1-60327-815-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation