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. 1982 Feb;79(4):988–992. doi: 10.1073/pnas.79.4.988

Purification and properties of the uvrA protein from Escherichia coli.

E Seeberg, A L Steinum
PMCID: PMC345884  PMID: 6280177

Abstract

The uvrA+ gene product from Escherichia coli was purified to apparent homogeneity; the assay measured its ability to restore repair endonuclease activity in extracts from uvrA mutated cells. The uvrA protein is a 115,000 molecular weight DNA-binding protein having higher affinity for single-stranded than double-stranded DNA. It does not introduce single-strand breaks or alkali-labile bonds in native or UV-irradiated DNA, but it catalyzes hydrolysis of ATP to ADP and Pi. The ATPase activity is not DNA dependent and has a Km of 0.23 mM, which corresponds to the Km for the ATP requirement of the UV-endonuclease reaction catalyzed by the combined uvrA+, uvrB+, and uvrC+ gene products. ADP and adenosine 5'-[gamma-thio]triphosphate both inhibit the uvrA ATPase as well as the uvrABC endonuclease and also prevent specific binding of the uvrA proteins to UV-irradiated DNA. These results indicate that both the DNA-binding property and the ATPase activity of the uvrA protein are essential for uvrABC endonuclease activity and that the ATP requirement of the endonuclease reaction is determined by uvrA ATPase.

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Selected References

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

  1. BOYCE R. P., HOWARD-FLANDERS P. RELEASE OF ULTRAVIOLET LIGHT-INDUCED THYMINE DIMERS FROM DNA IN E. COLI K-12. Proc Natl Acad Sci U S A. 1964 Feb;51:293–300. doi: 10.1073/pnas.51.2.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Cole R. S., Levitan D., Sinden R. R. Removal of psoralen interstrand cross-links from DNA of Escherichia coli: mechanism and genetic control. J Mol Biol. 1976 May 5;103(1):39–59. doi: 10.1016/0022-2836(76)90051-6. [DOI] [PubMed] [Google Scholar]
  4. Hanawalt P. C., Cooper P. K., Ganesan A. K., Smith C. A. DNA repair in bacteria and mammalian cells. Annu Rev Biochem. 1979;48:783–836. doi: 10.1146/annurev.bi.48.070179.004031. [DOI] [PubMed] [Google Scholar]
  5. Howard-Flanders P., Boyce R. P., Theriot L. Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics. 1966 Jun;53(6):1119–1136. doi: 10.1093/genetics/53.6.1119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ikenaga M., Ichikawa-Ryo H., Kondo S. The major cause of inactivation and mutation by 4-nitroquinoline 1-oixde in Escherichia coli: excisable 4NQO-purine adducts. J Mol Biol. 1975 Feb 25;92(2):341–356. doi: 10.1016/0022-2836(75)90233-8. [DOI] [PubMed] [Google Scholar]
  7. Kacinski B. M., Sancar A., Rupp W. D. A general approach for purifying proteins encoded by cloned genes without using a functional assay: isolation of the uvrA gene product from radiolabeled maxicells. Nucleic Acids Res. 1981 Sep 25;9(18):4495–4508. doi: 10.1093/nar/9.18.4495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kenyon C. J., Walker G. C. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci U S A. 1980 May;77(5):2819–2823. doi: 10.1073/pnas.77.5.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lawley P. D., Brookes P. Molecular mechanism of the cytotoxic action of difunctional alkylating agents and of resistance to this action. Nature. 1965 May 1;206(983):480–483. doi: 10.1038/206480a0. [DOI] [PubMed] [Google Scholar]
  10. Lindahl T. DNA glycosylases, endonucleases for apurinic/apyrimidinic sites, and base excision-repair. Prog Nucleic Acid Res Mol Biol. 1979;22:135–192. doi: 10.1016/s0079-6603(08)60800-4. [DOI] [PubMed] [Google Scholar]
  11. Ogawa T., Wabiko H., Tsurimoto T., Horii T., Masukata H., Ogawa H. Characteristics of purified recA protein and the regulation of its synthesis in vivo. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):909–915. doi: 10.1101/sqb.1979.043.01.099. [DOI] [PubMed] [Google Scholar]
  12. Roberts J. W., Roberts C. W., Craig N. L., Phizicky E. M. Activity of the Escherichia coli recA-gene product. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):917–920. doi: 10.1101/sqb.1979.043.01.100. [DOI] [PubMed] [Google Scholar]
  13. SETLOW R. B., CARRIER W. L. THE DISAPPEARANCE OF THYMINE DIMERS FROM DNA: AN ERROR-CORRECTING MECHANISM. Proc Natl Acad Sci U S A. 1964 Feb;51:226–231. doi: 10.1073/pnas.51.2.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sancar A., Wharton R. P., Seltzer S., Kacinski B. M., Clarke N. D., Rupp W. D. Identification of the uvrA gene product. J Mol Biol. 1981 May 5;148(1):45–62. doi: 10.1016/0022-2836(81)90234-5. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Seeberg E., Nissen-Meyer J., Strike P. Incision of ultraviolet-irradiated DNA by extracts of E. coli requires three different gene products. Nature. 1976 Oct 7;263(5577):524–526. doi: 10.1038/263524a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Seeberg E. Strand cleavage at psoralen adducts and pyrimidine dimers in DNA caused by interaction between semi-purified uvr+ gene products from Escherichia coli. Mutat Res. 1981 Jun;82(1):11–22. doi: 10.1016/0027-5107(81)90133-0. [DOI] [PubMed] [Google Scholar]
  19. Sugino A., Higgins N. P., Brown P. O., Peebles C. L., Cozzarelli N. R. Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4838–4842. doi: 10.1073/pnas.75.10.4838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Venitt S., Tarmy E. M. The selective excision of arylalkylated products from the DNA of Escherichia coli treated with the carcinogen 7-bromomethylbenz(a)anthracene. Biochim Biophys Acta. 1972 Nov 16;287(1):38–51. doi: 10.1016/0005-2787(72)90328-0. [DOI] [PubMed] [Google Scholar]
  21. Waldstein E. A., Sharon R., Ben-Ishai R. Role of ATP in excision repair of ultraviolet radiation damage in Escherichia coli. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2651–2654. doi: 10.1073/pnas.71.7.2651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weinstock G. M., McEntee K., Lehman I. R. ATP-dependent renaturation of DNA catalyzed by the recA protein of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jan;76(1):126–130. doi: 10.1073/pnas.76.1.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wickner S., Hurwitz J. Interaction of Escherichia coli dnaB and dnaC(D) gene products in vitro. Proc Natl Acad Sci U S A. 1975 Mar;72(3):921–925. doi: 10.1073/pnas.72.3.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. van de Putte P., van Sluis C. A., van Dillewijn J., Rörsch A. The location of genes controlling radiation sensitivity in Escherichia coli. Mutat Res. 1965 Apr;2(2):97–110. doi: 10.1016/0027-5107(65)90041-2. [DOI] [PubMed] [Google Scholar]

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