Mapping and Quantification of Bulky-Chemical-Induced DNA Damage Using UvrABC Nucleases

  • Moon-shong Tang

Abstract

Nucleotide excision repair (NER) is one of the most versatile and conservative repair systems in the biological kingdom. This repair pathway repairs DNA damage caused by a variety of agents, including ultraviolet light (UV), benzo[a]pyrene diol epoxide (BPDE), N-acetoxy-2-acetyl-aminofluorene (NAAAF), N-hydroxy-2-aminofluorene (N-OH-AF), dimethylbenzanthracene diol epoxide (DMBA-DE), and therapeutic drugs (cis-platinum, CC-1065, anthramycin, mitomycin C., and psoralen) in both prokaryotes and eukaryotes (for a review see Friedberg et al., 1995; Sancar and Tang, 1993; van Houten, 1990). The initial step of NER involves the recognition of the damaged bases and dual incisions at the 5′ and 3′ sides of the damaged base(s). In E. coli cells these steps are controlled by three gene products—the UvrA, UvrB, and UvrC proteins. The uvrA, uvrB, and uvrC genes have been cloned into expression vectors allowing relatively large quantities of these proteins to be readily purified without elaborate procedures. Consequently, the biochemical nature of the recognition of DNA damage and of the dual incisions 5′ and 3′ of the damaged base(s) by the coordinative mechanisms of these three proteins have been extensively studied. The details of these reactions can be found in several review articles (Sancar, 1994; Sancar and Tang, 1993; Grossman and Yeung, 1990; van Houten, 1990), and the following four steps represent a brief synopsis of the reactions of the Uvr proteins:
  1. 1.
    2 UvrA → (UvrA)2
    • In solution two UvrA proteins may form a dimer.

     
  2. 2.
    (UvrA)2 + UvrB → (UvrA)2·UvrB
    • The dimeric form of UvrA may bind to damaged DNA or form a complex with UvrB.

     
  3. 3.

    (UvrA)2·UvrB locates and binds to the damaged base(s) and UvrA is released from the complex.

     
  4. 4.

    UvrC joins the UvrB-damaged base(s) complex, resulting in 5′ and 3′ incisions.

     

Keywords

Vortex EDTA Electrophoresis Pyrene Borate 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Brash, D. E., and Haseltine, W. A. (1982). UV-induced mutation hotspots occur at DNA damage hotspots. Nature 298:189–192.PubMedCrossRefGoogle Scholar
  2. Carson, P. R., and Grossman, L. (1988). Potential role of proteolysis in the control of UvrABC incision. Nucleic Acids Res. 16:10903–10912.CrossRefGoogle Scholar
  3. Chen, J., Pao, A., Yi, Z., Ye, X., Morris, R., Harvey, R., and Tang, M.-s. (1996). Sequence preference of dimethylbenzanthracene diol epoxide-DNA binding in mouse H-ras detected by UvrABC nuclease. Submitted for publication.Google Scholar
  4. Chen, R.-H., Maher, V. M., Bouwer, J., van de Putte, P., and McCormick, J. J. (1992). Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in the HPRT gene of diploid human fibroblasts. Proc. Natl. Acad. Sci. USA 89:5413–5417.PubMedCrossRefGoogle Scholar
  5. Christner, D. R., Lakshman, M. K., Sayer, J. M., Jerina, D. M., and Dipple, A. (1994). Primer extension by various polymerase using oligonucleotide templates containing stereoisomeric benzo[a]pyrene-deoxyadenosine adducts. Biochemistry 33:14297–14305.PubMedCrossRefGoogle Scholar
  6. Doisy, R., and Tang, M.-s. (1995). Effect of aminofluorene and (acetylamino)fluorene adducts on the DNA replication mediated by Escherichia coli polymerase I (Klenow fragment) and III. Biochemistry 34:4358–4368.PubMedCrossRefGoogle Scholar
  7. Franklin, W. A., Lo, K. M., and Haseltine, W. A. (1982). Alkaline lability of fluorescent photoproducts produced in ultraviolet light-irradiated DNA. J. Biol. Chem. 257:13535–13543.PubMedGoogle Scholar
  8. Friedberg, E. C., Walker, G. C., and Siede, W. (1995). DNA Repair and Mutagenesis, ASM Press, Washington, DC.Google Scholar
  9. Gordon, L. K., and Haseltine, W. A. (1982). Quantitation of cyclobutane pyrimidine dimer formation in double-and single-strand DNA fragments of defined sequence. Radiat. Res. 89:99–112.PubMedCrossRefGoogle Scholar
  10. Grossman, L., and Yeung, A. T. (1990). The UvrABC endonuclease system of Escherichia coli—A view from Baltimore. Mutat. Res. 236:213–221.PubMedCrossRefGoogle Scholar
  11. Hurley, L., Reynolds, V. L., Swenson, D. H., Petzold, G. L., and Scahill, T. A. (1984). Reaction of the antitumor antibiotic CC-1065 with DNA:Structure of a DNA adduct with DNA sequence specificity. Science 226:843–844.PubMedCrossRefGoogle Scholar
  12. Kohn, H., Li, V.-S., and Tang, M.-s. (1992). Recognition of mitomycin C-DNA monoadducts by UVRABC nuclease. J. Am. Chem. Soc. 114:5501–5509.CrossRefGoogle Scholar
  13. Li, V.-S., and Kohn, H. (1991). Studies on the bonding specificity for mitomycin C-DNA monoalkylation processes. J. Am. Chem. Soc. 113:275–283.CrossRefGoogle Scholar
  14. Li, V.-S., Choi, D., Tang, M.-s., and Kohn, H. (1995). Structural requirement for mitomycin C DNA bonding. Biochemisyry 34:7120–7126.CrossRefGoogle Scholar
  15. Maxam, A. M., and Gilbert, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65:499–560.PubMedCrossRefGoogle Scholar
  16. Nazimiec, M., Grossman, L., and Tang, M.-s. (1992). A comparison of the rates of reaction and function of UVRB in UVRBC-and UVRAB-mediated anthramycin-N2-guanine-DNA repair. J. Biol. Chem. 267:24716–24724.PubMedGoogle Scholar
  17. Pierce, J. R., Case, R., and Tang, M.-s. (1989). Recognition and repair of 2-aminofluorene-and 2-(acetylamino)-fluorene-DNA adducts by UVRABC nuclease. Biochemistry 28:5821–5826.PubMedCrossRefGoogle Scholar
  18. Pierce, J. R., Nazimiec, M., and Tang, M-s. (1993). Comparision of sequence preference of tomaymycin-and anthramycin-DNA bonding by exonuclease III and X exonuclease digestion and UvrABC nuclease incision analysis. Biochemistry 32:7069–7078.PubMedCrossRefGoogle Scholar
  19. Sancar, A. (1994). Mechanisms of DNA excision repair. Science 266:1954–1956.PubMedCrossRefGoogle Scholar
  20. Sancar, A., and Rupp, W. D. (1983). A novel repair enzyme: UVRABC excision nuclease of Escherichia coli: Cut a DNA strand on both sides of the damaged region. Cell 33:249–260.PubMedCrossRefGoogle Scholar
  21. Sancar, A., and Tang, M-s. (1993). Nucleotide excision repair. Photochem. Photobiol 57:905–921.PubMedCrossRefGoogle Scholar
  22. Selby, C. P., and Sancar, A. (1990). Structure and function of the (A)BC excinuclease of Escherichia coli. Mutat. Res. 236:203–211.PubMedCrossRefGoogle Scholar
  23. Tang, M.-s., Lee, C.-S., Doisy, R., Ross, L. Needham-VanDevanter, N., and Hurley, L. H. (1988). Recognition and repair of the CC-1065-(N3-adenine)-DNA adduct by the UVRABC nucleases. Biochem. 27:893–901.CrossRefGoogle Scholar
  24. Tang, M.-s., Bohr, V. A., Zhang, X.-s., Pierce, J., and Hanawalt, P.C. (1989). Quantification of aminofluorene adduct formation and repair in defined DNA sequences in mammalian cells using the UVRABC nuclease. J. Biol. Chem. 264:14455–14462.PubMedGoogle Scholar
  25. Tang, M.-s., Htun, H., Cheng, Y., and Dahlberg, J. E. (1991). Suppression of cyclobutane and 6–4 dipyrimidines formation in triple-stranded H-DNA. Biochemistry 30:7021–7026.PubMedCrossRefGoogle Scholar
  26. Tang, M.-s., Pierce, J. R., Doisy, R. P., Nazimiec, M. E., and MacLeod M. C. (1992). Differences and similarities in the repair of two benzo[a]pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB, and uvrC gene products. Biochemistry 31:8429–8436.PubMedCrossRefGoogle Scholar
  27. Tang, M.-s., Pao, A., and Zhang, X.-s. (1994a). Repair of benzo(a)pyrene diol epoxide-and UV-induced DNA damage in dihydrofolate reductase and adenine phosphoribosyltransferase genes of CHO cells. J. Biol. Chem. 269:12749–12754.PubMedGoogle Scholar
  28. Tang, M.-s., Qian, M., and Pao, A. (1994b). Formation and repair of antitumor antibiotic CC-1065 induced DNA adducts in the adenine phosphoribosyltransferase and amplified dihydrofolate reductase genes of Chinese hamster ovary cells. Biochemistry 33:2726–2732.PubMedCrossRefGoogle Scholar
  29. Thomas, D. C., Levy, M., and Sancar, A. (1985). Amplification and purfication of UvrA, UvrB, and UvrC proteins of Escherichia coli. J. Biol. Chem. 260:9875–9883.PubMedGoogle Scholar
  30. Thomas, D. C., Morton, A. G., Bohr, V. A., and Sancar, A. (1988). General method for quantifying base adducts in specific mammalian genes. Proc. Natl. Acad. Sci. USA 85:3723–3727.PubMedCrossRefGoogle Scholar
  31. van Houten, B. (1990). Nucleotide excision repair in Escherichia coli. Microbiol. Rev. 45:18–51.Google Scholar
  32. Vreeswijk, M. P. G., van Hoffen, A., Westland, B. E., Vrieling, H., van Zeeland, A. A., and Mullenders, H. F. (1994). Analysis of repair of cyclobutane pyrimidine dimers and pyrimidine 6–4 pyrimidone photoproducts in transcriptionally active and inactive genes in Chinese hamster cells. J. Biol. Chem. 269:31858–31863.PubMedGoogle Scholar
  33. Walter, R. B., Pierce, J., Case, R., and Tang, M.-s. (1988). Recognition of the DNA helix stabilizing anthramycin-N2 guanine adduct by UVRABC nuclease. J. Mol. Biol. 203:939–947.PubMedCrossRefGoogle Scholar
  34. Yeung, A. T., Mattes, W. B., and Grossman, L. (1986). Protein complexes formed during the incision reaction catalyzed by the E. coli UvrABC endonuclease. Nucleic Acids Res. 14:2567–2582.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • Moon-shong Tang
    • 1
  1. 1.Department of CarcinogenesisUniversity of Texas M. D. Anderson Cancer CenterSmithvilleUSA

Personalised recommendations