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RecA acts in trans to allow replication of damaged DNA by DNA polymerase V

Abstract

The DNA polymerase V (pol V) and RecA proteins are essential components of a mutagenic translesion synthesis pathway in Escherichia coli designed to cope with DNA damage. Previously, it has been assumed that RecA binds to the DNA template strand being copied. Here we show, however, that pol-V-catalysed translesion synthesis, in the presence or absence of the β-processivity-clamp, occurs only when RecA nucleoprotein filaments assemble or RecA protomers bind on separate single-stranded (ss)DNA molecules in trans. A 3′-proximal RecA filament end on trans DNA is essential for stimulation; however, synthesis is strengthened by further pol V–RecA interactions occurring elsewhere along a trans nucleoprotein filament. We suggest that trans-stimulation of pol V by RecA bound to ssDNA reflects a distinctive regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis on a damaged template strand obstructing translesion DNA synthesis despite the absolute requirement of RecA for SOS mutagenesis.

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Figure 1: RecA–ssDNA transactivation of pol V on damaged and undamaged DNA templates.
Figure 2: Kinetics of pol V transactivation by RecA–ssDNA.
Figure 3: A RecA filament end oriented 3′ on trans DNA is required for efficient stimulation of pol V.
Figure 4: Models depicting RecA–DNA transactivation of pol-V-catalysed translesion DNA synthesis.

References

  1. Radman, M. in Molecular and Environmental Aspects of Mutagenesis (eds Prakash, L., Sherman, F., Miller, M. W., Lawrence, C. W. & Tabor, H. W.) 128–142 (Charles C. Thomas, Springfield, Illinois, 1974)

    Google Scholar 

  2. Witkin, E. M. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40, 869–907 (1976)

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Friedberg, E. C. et al. DNA Repair and Mutagenesis 2nd edn, 461–555 (ASM Press, Washington DC, 2006)

    Google Scholar 

  4. Goodman, M. F. Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu. Rev. Biochem. 71, 17–50 (2002)

    CAS  Article  Google Scholar 

  5. 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)

    ADS  CAS  Article  Google Scholar 

  6. 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)

    ADS  CAS  Article  Google Scholar 

  7. 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)

    CAS  Article  Google Scholar 

  8. 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)

    CAS  Article  Google Scholar 

  9. 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)

    ADS  CAS  Article  Google Scholar 

  10. 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)

    CAS  Article  Google Scholar 

  11. 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)

    CAS  Article  Google Scholar 

  12. Pham, P. et al. A model for SOS-lesion targeted mutations in E. coli involving pol V, RecA, SSB and β sliding clamp. Nature 409, 366–370 (2001)

    ADS  CAS  Article  Google Scholar 

  13. Reuven, N. B. et al. Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament. J. Biol. Chem. 276, 5511–5517 (2001)

    CAS  Article  Google Scholar 

  14. 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)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  17. Pham, P. et al. Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis. Proc. Natl Acad. Sci. USA 99, 11061–11066 (2002)

    ADS  CAS  Article  Google Scholar 

  18. Maor-Shoshani, A., Reuven, N. B., Tomer, G. & Livneh, Z. Highly mutagenic replication by DNA polymerase V (UmuC) provides a mechanistic basis for SOS untargeted mutagenesis. Proc. Natl Acad. Sci. USA 97, 565–570 (2000)

    ADS  CAS  Article  Google Scholar 

  19. Fujii, S. & Fuchs, R. P. Defining the position of the switches between replicative and bypass DNA polymerases. EMBO J. 23, 4342–4352 (2004)

    CAS  Article  Google Scholar 

  20. 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 

  21. Stohl, E. A. et al. Escherichia coli RecX inhibits RecA recombinase and coprotease activities in vitro and in vivo. J. Biol. Chem. 278, 2278–2285 (2003)

    CAS  Article  Google Scholar 

  22. Drees, J. C. et al. A RecA filament capping mechanism for RecX protein. Mol. Cell 15, 789–798 (2004)

    CAS  Article  Google Scholar 

  23. Bailone, A. et al. A RecA protein mutant deficient in its interaction with the UmuD' C complex. Biochemie 73, 479–484 (1991)

    CAS  Article  Google Scholar 

  24. 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)

    CAS  Article  Google Scholar 

  25. Lavery, P. E. & Kowalczykowski, S. C. Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins. J. Biol. Chem. 267, 20648–20658 (1992)

    CAS  PubMed  Google Scholar 

  26. VanLoock, M. S. et al. ATP-mediated conformational changes in the RecA filament. Structure 11, 187–196 (2003)

    CAS  Article  Google Scholar 

  27. Frank, E. G. et al. Visualization of two binding sites for the Escherichia coli UmuD′2C complex (DNA pol V) on RecA–ssDNA filaments. J. Mol. Biol. 297, 585–597 (2000)

    CAS  Article  Google Scholar 

  28. Boudsocq, F., Campbell, M., Devoret, R. & Bailone, A. Quantitation of the inhibition of Hfr × F- recombination by the mutagenesis complex UmuD' C. J. Mol. Biol. 270, 201–211 (1997)

    CAS  Article  Google Scholar 

  29. Sommer, S., Bailone, A. & Devoret, R. The appearance of the UmuD' C protein complex in Escherichia coli switches repair from homologous recombination to SOS mutagenesis. Mol. Microbiol. 10, 963–971 (1993)

    CAS  Article  Google Scholar 

  30. Maor-Shoshani, A. M. & Livneh, Z. Analysis of the stimulation of DNA polymerase V of Escherichia coli by processivity proteins. Biochemistry 41, 14438–14446 (2002)

    CAS  Article  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by National Institutes of Health grants to M.F.G. and M.M.C., and funds from the NICHD/NIH Intramural Research Program to R.W.

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Correspondence to Myron F. Goodman.

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

Supplementary information

Supplementary Figures

This file contains a background figure (Supplementary Figure 1), supportive data (Supplementary Figures 2–12) and DNA sequences (Figure 13). (PDF 1094 kb)

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This file contains additional details of the Methods used in this study. (PDF 56 kb)

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Schlacher, K., Cox, M., Woodgate, R. et al. RecA acts in trans to allow replication of damaged DNA by DNA polymerase V. Nature 442, 883–887 (2006). https://doi.org/10.1038/nature05042

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