Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The active form of DNA polymerase V is UmuD′2C–RecA–ATP

Nature volume 460, pages 359–363 (2009)Cite this article

Abstract

DNA-damage-induced SOS mutations arise when Escherichia coli DNA polymerase (pol) V, activated by a RecA nucleoprotein filament (RecA*), catalyses translesion DNA synthesis. Here we address two longstanding enigmatic aspects of SOS mutagenesis, the molecular composition of mutagenically active pol V and the role of RecA*. We show that RecA* transfers a single RecA–ATP stoichiometrically from its DNA 3′-end to free pol V (UmuD′2C) to form an active mutasome (pol V Mut) with the composition UmuD′2C–RecA–ATP. Pol V Mut catalyses TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecA–ATP in the complex. Reactivation of pol V Mut is triggered by replacement of RecA–ATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecA–ATP to pol V, and thus generate active mutasomal complex for translesion synthesis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: DNA synthesis by pol V Mut or pol V transactivated by RecA*.
Figure 2: Activated pol V Mut complex is composed of UmuD′ 2 C–RecA–ATP.
Figure 3: Determination of the molecular mass of pol V Mut–(wild-type RecA) by MALS.
Figure 4: Pol V activation requires transfer of a RecA molecule from the DNA 3′ end of RecA*.
Figure 5: Model depicting pol V activation, deactivation and reactivation.

Similar content being viewed by others

References

  1. Tang, M. J. et al. UmuD′2C is an error-prone DNA polymerase, Escherichia coli pol V. Proc. Natl Acad. Sci. USA 96, 8919–8924 (1999)

    Article  ADS  CAS  Google Scholar 

  2. Reuven, N. B., Arad, G., Maor-Shoshani, A. & Livneh, Z. The mutagenesis protein UmuC is a DNA polymerase activated by UmuD′, RecA, and SSB and is specialized for translesion replication. J. Biol. Chem. 274, 31763–31766 (1999)

    Article  CAS  Google Scholar 

  3. Friedberg, E. C. et al. DNA Repair and Mutagenesis 2nd edn 463–555 (ASM Press, 2006)

    Google Scholar 

  4. Pham, P., Bertram, J. G., O'Donnell, M., Woodgate, R. & Goodman, M. F. A model for SOS-lesion-targeted mutations in Escherichia coli. Nature 409, 366–370 (2001)

    Article  ADS  CAS  Google Scholar 

  5. Becherel, O. J., Fuchs, R. P. & Wagner, J. Pivotal role of the β-clamp in translesion DNA synthesis and mutagenesis in E. coli cells. DNA Repair 1, 703–708 (2002)

    Article  CAS  Google Scholar 

  6. Lenne-Samuel, N., Wagner, J., Etienne, H. & Fuchs, R. P. The processivity factor β controls DNA polymerase IV traffic during spontaneous mutagenesis and translesion synthesis in vivo. EMBO Rep. 3, 45–49 (2002)

    Article  CAS  Google Scholar 

  7. Tang, M. et al. Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis. Nature 404, 1014–1018 (2000)

    Article  ADS  CAS  Google Scholar 

  8. Kato, T. & Shinoura, Y. Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol. Gen. Genet. 156, 121–131 (1977)

    CAS  PubMed  Google Scholar 

  9. Steinborn, G. Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis. I. Isolation of uvm mutants and their phenotypical characterization in DNA repair and mutagenesis. Mol. Gen. Genet. 165, 87–93 (1978)

    Article  CAS  Google Scholar 

  10. Bruck, I., Woodgate, R., McEntee, K. & Goodman, M. F. Purification of a soluble UmuD′C complex from Escherichia coli: cooperative binding of UmuD′C to single-stranded DNA. J. Biol. Chem. 271, 10767–10774 (1996)

    Article  CAS  Google Scholar 

  11. Woodgate, R., Rajagopalan, M., Lu, C. & Echols, H. UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD′. Proc. Natl Acad. Sci. USA 86, 7301–7305 (1989)

    Article  ADS  CAS  Google Scholar 

  12. Tang, M. et al. Biochemical basis of SOS-induced mutagenesis in Escherichia coli: reconstitution of in vitro lesion bypass dependent on the UmuD′2C mutagenic complex and RecA protein. Proc. Natl Acad. Sci. USA 95, 9755–9760 (1998)

    Article  ADS  CAS  Google Scholar 

  13. Fujii, S., Gasser, V. & Fuchs, R. P. The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited. J. Mol. Biol. 341, 405–417 (2004)

    Article  CAS  Google Scholar 

  14. Nohmi, T., Battista, J. R., Dodson, L. A. & Walker, G. C. RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc. Natl Acad. Sci. USA 85, 1816–1820 (1988)

    Article  ADS  CAS  Google Scholar 

  15. Dutreix, M. et al. New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis. J. Bacteriol. 171, 2415–2423 (1989)

    Article  CAS  Google Scholar 

  16. Sweasy, J. B., Witkin, E. M., Sinha, N. & Roegner-Maniscalco, V. RecA protein of Escherichia coli has a third essential role in SOS mutator activity. J. Bacteriol. 172, 3030–3036 (1990)

    Article  CAS  Google Scholar 

  17. Kuzminov, A. Recombinational repair of DNA damage in Escherichia coli and bacteriophage λ. Microbiol. Mol. Biol. Rev. 63, 751–813 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Bridges, B. A. & Woodgate, R. Mutagenic repair in Escherichia coli. X. The umuC gene product may be required for replication past pyrimidine dimers but not for the coding error in UV-mutagenesis. Mol. Gen. Genet. 196, 364–366 (1984)

    Article  CAS  Google Scholar 

  19. Echols, H. & Goodman, M. F. Mutation induced by DNA damage: a many protein affair. Mutat. Res. 236, 301–311 (1990)

    Article  CAS  Google Scholar 

  20. Sommer, S., Boudsocq, F., Devoret, R. & Bailone, A. Specific RecA amino acid changes affect RecA–UmuD′C interaction. Mol. Microbiol. 28, 281–291 (1998)

    Article  CAS  Google Scholar 

  21. Schlacher, K., Cox, M. M., Woodgate, R. & Goodman, M. F. RecA acts in trans to allow replication of damaged DNA by DNA polymerase V. Nature 442, 883–887 (2006)

    Article  ADS  CAS  Google Scholar 

  22. Eggler, A. L., Lusetti, S. L. & Cox, M. M. The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein. J. Biol. Chem. 278, 16389–16396 (2003)

    Article  CAS  Google Scholar 

  23. Dutreix, M., Burnett, B., Bailone, A., Radding, C. M. & Devoret, R. A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments. Mol. Gen. Genet. 232, 489–497 (1992)

    Article  CAS  Google Scholar 

  24. Wyatt, P. J. Light scattering and the absolute characterization of macromolecules. Anal. Chim. Acta 272, 1–40 (1993)

    Article  CAS  Google Scholar 

  25. Schlacher, K. et al. DNA polymerase V and RecA protein, a minimal mutasome. Mol. Cell 17, 561–572 (2005)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by National Institutes of Health grants to M.F.G. (ES12259; R37GM21422) and M.M.C. (GM32335), and funds from the NICHD/NIH Intramural Research Program to K.K. and R.W. The authors thank R. Britt for preparation of the RecA E38KΔC17 protein used in this study. MALS data were collected using the USC NanoBiophysics Core Facility, with the aid and cooperation of N. Chelyapov. We thank J. Bertram for his help in performing the MALS experiment and analysing the data. We thank J. Petruska for his comments.

Author Contributions Q.J. performed the experiments and experimental analyses; K.K. performed the construction and purification of His-tagged pol V; M.M.C. provided the purified RecA E38KΔC17 protein; Q.J. and M.F.G. designed the experiments with input from M.M.C. and R.W.; M.F.G., Q.J., M.M.C. and R.W. wrote the manuscript.

Author information

Authors and Affiliations

  1. Departments of Biological Sciences and Chemistry, University of Southern California, University Park, Los Angeles, California 90089-2910, USA,

    Qingfei Jiang & Myron F. Goodman

  2. Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-3371, USA,

    Kiyonobu Karata & Roger Woodgate

  3. Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA,

    Michael M. Cox

Authors
  1. Qingfei Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kiyonobu Karata
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roger Woodgate
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael M. Cox
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Myron F. Goodman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Myron F. Goodman.

Supplementary information

Supplementary Information

This file contains Supplementary Methods and Supplementary Figures 1-9. Supplementary Figures were corrected on 23 July, 2009. (PDF 1379 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jiang, Q., Karata, K., Woodgate, R. et al. The active form of DNA polymerase V is UmuD′2C–RecA–ATP. Nature 460, 359–363 (2009). https://doi.org/10.1038/nature08178

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08178

This article is cited by

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

Advanced search

Quick links

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