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A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates

Nature volume 439, pages 225–228 (2006)Cite this article

Abstract

Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance1. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase η (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD′2C), respectively2,3. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic4. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase κ, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10–15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the ‘steric gate’ residue of DinB (Phe13 → Val) and that of its archaeal homologue Dbh (Phe12 → Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.

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Figure 3: Importance of DinB F13 residue in vivo.
Figure 1: Bypass of N 2 -furfuryl-dG.
Figure 2: A single mutation in DinB or its archaeal orthologue Dbh separates their TLS and DNA polymerase activities.

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References

  1. Friedberg, E. C., Wagner, R. & Radman, M. Specialized DNA polymerases, cellular survival, and the genesis of mutations. Science 296, 1627–1630 (2002)

    Article  CAS  ADS  Google Scholar 

  2. Washington, M. T., Johnson, R. E., Prakash, L. & Prakash, S. Accuracy of lesion bypass by yeast and human DNA polymerase η. Proc. Natl Acad. Sci. USA 98, 8355–8360 (2001)

    Article  CAS  ADS  Google Scholar 

  3. 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  CAS  ADS  Google Scholar 

  4. Fuchs, R. P., Fujii, S. & Wagner, J. Properties and functions of Escherichia coli: pol IV and pol V. Adv. Protein Chem. 69, 229–264 (2004)

    Article  CAS  Google Scholar 

  5. Kenyon, C. J. & Walker, G. C. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc. Natl Acad. Sci. USA 77, 2819–2823 (1980)

    Article  CAS  ADS  Google Scholar 

  6. Brotcorne-Lannoye, A. & Maenhaut-Michel, G. Role of RecA protein in untargeted UV mutagenesis of bacteriophage λ: evidence for the requirement for the dinB gene. Proc. Natl Acad. Sci. USA 83, 3904–3908 (1986)

    Article  CAS  ADS  Google Scholar 

  7. McKenzie, G. J., Lee, P. L., Lombardo, M. J., Hastings, P. J. & Rosenberg, S. M. SOS mutator DNA polymerase IV functions in adaptive mutation and not adaptive amplification. Mol. Cell 7, 571–579 (2001)

    Article  CAS  Google Scholar 

  8. Kim, S. R. et al. Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA. Proc. Natl Acad. Sci. USA 94, 13792–13797 (1997)

    Article  CAS  ADS  Google Scholar 

  9. Kim, S. R., Matsui, K., Yamada, M., Gruz, P. & Nohmi, T. Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli. Mol. Genet. Genomics 266, 207–215 (2001)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Whiteway, J. et al. Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli. J. Bacteriol. 180, 5529–5539 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Panigrahi, G. B. & Walker, I. G. The N2-guanine adduct but not the C8-guanine or N6-adenine adducts formed by 4-nitroquinoline 1-oxide blocks the 3′–5′ exonuclease action of T4 DNA polymerase. Biochemistry 29, 2122–2126 (1990)

    Article  CAS  Google Scholar 

  13. Creighton, S., Bloom, L. B. & Goodman, M. F. Gel fidelity assay measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies. Methods Enzymol. 262, 232–256 (1995)

    Article  CAS  Google Scholar 

  14. Schwede, T., Kopp, J., Guex, N. & Peitsch, M. C. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385 (2003)

    Article  CAS  Google Scholar 

  15. Ling, H., Boudsocq, F., Woodgate, R. & Yang, W. Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell 107, 91–102 (2001)

    Article  CAS  Google Scholar 

  16. DeLucia, A. M., Grindley, N. D. & Joyce, C. M. An error-prone family Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a ‘steric gate’ residue for discrimination against ribonucleotides. Nucleic Acids Res. 31, 4129–4137 (2003)

    Article  CAS  Google Scholar 

  17. Shen, X. et al. Efficiency and accuracy of SOS-induced DNA polymerases replicating benzo[a]pyrene-7,8-diol 9,10-epoxide A and G adducts. J. Biol. Chem. 277, 5265–5274 (2002)

    Article  CAS  Google Scholar 

  18. Gruz, P. et al. Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C. J. Biol. Chem. 276, 47394–47401 (2001)

    Article  CAS  Google Scholar 

  19. Wagner, J. et al. The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis. Mol. Cell 4, 281–286 (1999)

    Article  CAS  Google Scholar 

  20. Rydberg, B. & Game, J. Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts. Proc. Natl Acad. Sci. USA 99, 16654–16659 (2002)

    Article  CAS  ADS  Google Scholar 

  21. Watanabe, M., Nohmi, T. & Ohta, T. Effects of the umuDC, mucAB, and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: II. Base substitution mutagenesis. Mutat. Res. 314, 39–49 (1994)

    Article  CAS  Google Scholar 

  22. Maher, V. M., Ouellette, L. M., Curren, R. D. & McCormick, J. J. Frequency of ultraviolet light-induced mutations is higher in xeroderma pigmentosum variant cells than in normal human cells. Nature 261, 593–595 (1976)

    Article  CAS  ADS  Google Scholar 

  23. Cupples, C. G. & Miller, J. H. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc. Natl Acad. Sci. USA 86, 5345–5349 (1989)

    Article  CAS  ADS  Google Scholar 

  24. Astatke, M., Ng, K., Grindley, N. D. & Joyce, C. M. A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides. Proc. Natl Acad. Sci. USA 95, 3402–3407 (1998)

    Article  CAS  ADS  Google Scholar 

  25. Marnett, L. J. Oxy radicals, lipid peroxidation and DNA damage. Toxicology 181–182, 219–222 (2002)

    Article  Google Scholar 

  26. Zewail-Foote, M. et al. The inefficiency of incisions of ecteinascidin 743–DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent. Chem. Biol. 8, 1033–1049 (2001)

    Article  CAS  Google Scholar 

  27. Yasui, M. et al. Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells. Biochemistry 43, 15005–15013 (2004)

    Article  CAS  Google Scholar 

  28. DeCorte, B. L. et al. Improved strategies for postoligomerization synthesis of oligodeoxynucleotides bearing structurally defined adducts at the N2 position of deoxyguanosine. Chem. Res. Toxicol. 9, 630–637 (1996)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Joyce and A. DeLucia for Dbh and Dbh(F12A); E. Friedberg, J. Stirman and P. Fischhaber for Mus musculus pol κ; T. Nohmi for pYG768 and pYG782; N. Geacintov for benzo[a]pyrene-damaged substrate; A. Banerjee for assistance with Supplementary Fig. S5; and J. Tuttle and J. Bowers for advice on protein purification. This work was supported by the NIH (grants to G.C.W. and J.M.E.). Author Contributions D.F.J. performed the protein purification and lesion bypass assays, and proposed the involvement of the steric gate residue in TLS. V.G.G. discovered the sensitivity of a ΔdinB strain to NFZ and 4-NQO and performed the mutagenesis experiments. J.C.D. constructed and purified the N2-furfuryl-dG-containing oligonucleotide substrate.

Author information

Author notes

  1. Daniel F. Jarosz and Veronica G. Godoy: *These authors contributed equally to this work

Authors and Affiliations

  1. Department of Chemistry,

    Daniel F. Jarosz, James C. Delaney & John M. Essigmann

  2. Department of Biology,

    Veronica G. Godoy & Graham C. Walker

  3. Biological Engineering Division, Massachusetts Institute of Technology, Massachusetts, 02139, Cambridge, USA

    James C. Delaney & John M. Essigmann

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Correspondence to Graham C. Walker.

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Supplementary information

Supplementary Figure 1

Sensitivity of strains bearing a deletion of dinB to NFZ and 4-NQO. (PPT 1354 kb)

Supplementary Figure 2

SDS–PAGE of purified native DinB. (PPT 543 kb)

Supplementary Figure 3

Structures of lesions used in this study. (PPT 71 kb)

Supplementary Figure 4

Schematic of running start, standing start, and mismatch extension assays. (PPT 24 kb)

Supplementary Figure 5

A homology model of DinB, constructed in part with SWISS-Model, reveals an N2-dG lesion binding pocket and suggests a mechanism for rate enhancement on a damaged template. (PPT 1410 kb)

Supplementary Figure 6

SDS–PAGE of purified DinB(F13V). (PPT 373 kb)

Supplementary Figure 7

Products of running start primer extension reactions (30 min) with 1, 10, and 50 nM DinB and DinB(F13V) on undamaged and tetrahydrofuran bearing templates reveal that the F13V mutation does not render DinB able to bypass this lesion. (PPT 3828 kb)

Supplementary Figure 8

Dominant negativity of pdinB(F13V). (PPT 410 kb)

Supplementary Figure 9

MALDI-TOF mass spectrometry of the N2-furfuryl-dG adduct. (PPT 64 kb)

Supplementary Figure Legends

Text to accompany the above Supplementary Figures. (DOC 30 kb)

Supplementary Methods

This describes strain and plasmid construction, protein purification, and detailed oligonucleotide substrate synthesis methods. (DOC 25 kb)

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Jarosz, D., Godoy, V., Delaney, J. et al. A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates. Nature 439, 225–228 (2006). https://doi.org/10.1038/nature04318

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