Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Oct;80(20):6157–6161. doi: 10.1073/pnas.80.20.6157

Enzymatic properties of purified Escherichia coli uvrABC proteins.

A T Yeung, W B Mattes, E Y Oh, L Grossman
PMCID: PMC390162  PMID: 6312446

Abstract

The cloned uvrA and uvrB genes of Escherichia coli K-12 were amplified by linkage to the PL promoter of plasmid pKC30. The uvrC gene was amplified in the high-copy-number plasmid pRLM 24. The three gene products (purified in each case to greater than 95% purity) and ATP are required to effectively incise UV-damaged DNAs. The uvrABC proteins bind tightly to damaged sites in DNA, requiring the initial attachment of the uvrA protein in the presence of ATP before productive binding of the uvrB and uvrC proteins. Using a cloned tandem double insert of the lac p-o region as a damaged DNA substrate for the uvrABC complex and analyzing the incision both 5' and 3' to each pyrimidine dimer, we found that one break occurs 7 nucleotides 5' to a pyrimidine dimer and a second break is made 3-4 nucleotides 3' from the same pair of pyrimidines in the dimer. No such breaks are found in the strand complementary to the dimer. The size of the incised fragment in the DNA suggests that incision may be coordinated with excision reactions in repair processes.

Full text

PDF
6159

Images in this article

6158Image
on p.6158
6160Image
on p.6160
6160Image
on p.6160
6160Image
on p.6160

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. D'Andrea A. D., Haseltine W. A. Sequence specific cleavage of DNA by the antitumor antibiotics neocarzinostatin and bleomycin. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3608–3612. doi: 10.1073/pnas.75.8.3608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Demple B., Linn S. DNA N-glycosylases and UV repair. Nature. 1980 Sep 18;287(5779):203–208. doi: 10.1038/287203a0. [DOI] [PubMed] [Google Scholar]
  3. Grafstrom R. H., Park L., Grossman L. Enzymatic repair of pyrimidine dimer-containing DNA. A 5' dimer DNA glycosylase: 3'-apyrimidinic endonuclease mechanism from Micrococcus luteus. J Biol Chem. 1982 Nov 25;257(22):13465–13474. [PubMed] [Google Scholar]
  4. Grossman L., Riazuddin S., Haseltine W. A., Lindan C. Nucleotide excision repair of damaged DNA. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):947–955. doi: 10.1101/sqb.1979.043.01.104. [DOI] [PubMed] [Google Scholar]
  5. Haseltine W. A., Gordon L. K., Lindan C. P., Grafstrom R. H., Shaper N. L., Grossman L. Cleavage of pyrimidine dimers in specific DNA sequences by a pyrimidine dimer DNA-glycosylase of M. luteus. Nature. 1980 Jun 26;285(5767):634–641. doi: 10.1038/285634a0. [DOI] [PubMed] [Google Scholar]
  6. Johnsrud L. Contacts between Escherichia coli RNA polymerase and a lac operon promoter. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5314–5318. doi: 10.1073/pnas.75.11.5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kacinski B. M., Rupp W. D. E. coli uvrB protein binds to DNA in the presence of uvrA protein. Nature. 1981 Dec 3;294(5840):480–481. doi: 10.1038/294480a0. [DOI] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  10. McMillan S., Edenberg H. J., Radany E. H., Friedberg R. C., Friedberg E. C. den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities. J Virol. 1981 Oct;40(1):211–223. doi: 10.1128/jvi.40.1.211-223.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nakabeppu Y., Sekiguchi M. Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA. Proc Natl Acad Sci U S A. 1981 May;78(5):2742–2746. doi: 10.1073/pnas.78.5.2742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Neal M. W., Florini J. R. A rapid method for desalting small volumes of solution. Anal Biochem. 1973 Sep;55(1):328–330. doi: 10.1016/0003-2697(73)90325-4. [DOI] [PubMed] [Google Scholar]
  13. Radany E. H., Friedberg E. C. A pyrimidine dimer-DNA glycosylase activity associated with the v gene product of bacterophage T4. Nature. 1980 Jul 10;286(5769):182–185. doi: 10.1038/286182a0. [DOI] [PubMed] [Google Scholar]
  14. Riazuddin S., Grossman L. Micrococcus luteus correndonucleases. I. resolution and purification of two endonucleases specific for DNA containing pyrimidine dimers. J Biol Chem. 1977 Sep 25;252(18):6280–6286. [PubMed] [Google Scholar]
  15. Riggs A. D., Bourgeois S., Newby R. F., Cohn M. DNA binding of the lac repressor. J Mol Biol. 1968 Jul 14;34(2):365–368. doi: 10.1016/0022-2836(68)90261-1. [DOI] [PubMed] [Google Scholar]
  16. Rupp W. D., Sancar A., Sancar G. B. Properties and regulation of the UVRABC endonuclease. Biochimie. 1982 Aug-Sep;64(8-9):595–598. doi: 10.1016/s0300-9084(82)80094-1. [DOI] [PubMed] [Google Scholar]
  17. Sancar A., Rupp W. D. A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region. Cell. 1983 May;33(1):249–260. doi: 10.1016/0092-8674(83)90354-9. [DOI] [PubMed] [Google Scholar]
  18. Seeberg E. Multiprotein interactions in strand cleavage of DNA damaged by UV and chemicals. Prog Nucleic Acid Res Mol Biol. 1981;26:217–226. doi: 10.1016/s0079-6603(08)60406-7. [DOI] [PubMed] [Google Scholar]
  19. Seeberg E. Reconstitution of an Escherichia coli repair endonuclease activity from the separated uvrA+ and uvrB+/uvrC+ gene products. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2569–2573. doi: 10.1073/pnas.75.6.2569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Seeberg E., Steinum A. L. Purification and properties of the uvrA protein from Escherichia coli. Proc Natl Acad Sci U S A. 1982 Feb;79(4):988–992. doi: 10.1073/pnas.79.4.988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tanaka T., Weisblum B. Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J Bacteriol. 1975 Jan;121(1):354–362. doi: 10.1128/jb.121.1.354-362.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yoakum G. H., Kushner S. R., Grossman L. Isolation of plasmids carrying either the uvrC or uvrC uvrA and ssb genes of Escherichia coli K-12. Gene. 1980 Dec;12(3-4):243–248. doi: 10.1016/0378-1119(80)90106-7. [DOI] [PubMed] [Google Scholar]
  23. Yoakum G. H., Yeung A. T., Mattes W. B., Grossman L. Amplification of the uvrA gene product of Escherichia coli to 7% of cellular protein by linkage to the pL promoter of pKC30. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1766–1770. doi: 10.1073/pnas.79.6.1766. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES