Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Apr 11;11(7):2193–2204. doi: 10.1093/nar/11.7.2193

An in vitro complementation assay for the Escherichia coli uvrD gene product.

N B Kuemmerle, W E Masker
PMCID: PMC325872  PMID: 6300798

Abstract

An in vitro assay specific for the product of the uvrD gene of Escherichia coli has been developed. This assay, derived from properties of uvrD mutants revealed by in vivo experiments, is based on the necessity for a functional UvrD protein for complete rejoining of covalently closed circular DNA during the excision repair of UV-induced damage. Extracts prepared from gently lysed uvrD101 mutant cells are capable of restoring UV-damaged DNA to its covalently closed circular form when provided with a functional UvrD protein from other repair-deficient cell extracts or from partially purified protein fractions. This assay was employed to monitor the activity of the UvrD protein after several steps of fractionation. The partially purified UvrD protein does not complement extracts deficient in DNA polymerase I or temperature-sensitive in DNA ligase; it does, however, complement extracts from strains mutant at the uvrE and recL loci, which are considered allelic with the uvrD locus.

Full text

PDF
2197

Selected References

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

  1. Arthur H. M., Lloyd R. G. Hyper-recombination in uvrD mutants of Escherichia coli K-12. Mol Gen Genet. 1980;180(1):185–191. doi: 10.1007/BF00267368. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Carlson K. M., Smith K. C. Effect of the uvrD3 mutation on ultraviolet radiation-induced DNA-repair replication in Escherichia coli K12. Mutat Res. 1981 Dec;84(2):257–262. doi: 10.1016/0027-5107(81)90195-0. [DOI] [PubMed] [Google Scholar]
  4. Espejo R. T., Canelo E. S. Properties of bacteriophage PM2: a lipid-containing bacterial virus. Virology. 1968 Apr;34(4):738–747. doi: 10.1016/0042-6822(68)90094-9. [DOI] [PubMed] [Google Scholar]
  5. Gottesman M. M., Hicks M. L., Gellert M. Genetics and function of DNA ligase in Escherichia coli. J Mol Biol. 1973 Jul 15;77(4):531–547. doi: 10.1016/0022-2836(73)90221-0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Kanner L., Hanawalt P. Repair deficiency in a bacterial mutant defective in DNA polymerase. Biochem Biophys Res Commun. 1970 Apr 8;39(1):149–155. doi: 10.1016/0006-291x(70)90770-9. [DOI] [PubMed] [Google Scholar]
  8. Kenyon C. J., Walker G. C. Expression of the E. coli uvrA gene is inducible. Nature. 1981 Feb 26;289(5800):808–810. doi: 10.1038/289808a0. [DOI] [PubMed] [Google Scholar]
  9. Kuemmerle N. B., Ley R. D., Masker W. E. The effect of mutations in the uvrD cistron of Escherichia coli on repair resynthesis. Mutat Res. 1982 Jun;94(2):285–297. doi: 10.1016/0027-5107(82)90292-5. [DOI] [PubMed] [Google Scholar]
  10. Kuemmerle N. B., Masker W. E. Effect of the uvrD mutation on excision repair. J Bacteriol. 1980 May;142(2):535–546. doi: 10.1128/jb.142.2.535-546.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kuemmerle N. B., Masker W. E. In vitro packaging of UV radiation-damaged DNA from bacteriophage T7. J Virol. 1977 Sep;23(3):509–516. doi: 10.1128/jvi.23.3.509-516.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Litman R. M. A deoxyribonucleic acid polymerase from Micrococcus luteus (Micrococcus lysodeikticus) isolated on deoxyribonucleic acid-cellulose. J Biol Chem. 1968 Dec 10;243(23):6222–6233. [PubMed] [Google Scholar]
  14. Maples V. F., Kushner S. R. DNA repair in Escherichia coli: identification of the uvrD gene product. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5616–5620. doi: 10.1073/pnas.79.18.5616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nevers P., Spatz H. C. Escherichia coli mutants uvr D and uvr E deficient in gene conversion of lambda-heteroduplexes. Mol Gen Genet. 1975 Aug 27;139(3):233–243. doi: 10.1007/BF00268974. [DOI] [PubMed] [Google Scholar]
  16. Oeda K., Horiuchi T., Sekiguchi M. Molecular cloning of the uvrD gene of Escherichia coli that controls ultraviolet sensitivity and spontaneous mutation frequency. Mol Gen Genet. 1981;184(2):191–199. doi: 10.1007/BF00272904. [DOI] [PubMed] [Google Scholar]
  17. Oeda K., Horiuchi T., Sekiguchi M. The uvrD gene of E. coli encodes a DNA-dependent ATPase. Nature. 1982 Jul 1;298(5869):98–100. doi: 10.1038/298098a0. [DOI] [PubMed] [Google Scholar]
  18. Ogawa H., Shimada K., Tomizawa J. Studies on radiation-sensitive mutants of E. coli. I. Mutants defective in the repair synthesis. Mol Gen Genet. 1968 May 3;101(3):227–244. doi: 10.1007/BF00271625. [DOI] [PubMed] [Google Scholar]
  19. Rothman R. H., Clark A. J. Defective excision and postreplication repair of UV-damaged DNA in a recL mutant strain of E. coli K-12. Mol Gen Genet. 1977 Oct 24;155(3):267–277. doi: 10.1007/BF00272805. [DOI] [PubMed] [Google Scholar]
  20. Rothman R. H. Dimer excision and repair replication patch size in recL152 mutant of Escherichia coli K-12. J Bacteriol. 1978 Oct;136(1):444–448. doi: 10.1128/jb.136.1.444-448.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rydberg B. Bromouracil mutagenesis and mismatch repair in mutator strains of Escherichia coli. Mutat Res. 1978 Oct;52(1):11–24. doi: 10.1016/0027-5107(78)90091-x. [DOI] [PubMed] [Google Scholar]
  22. Sancar G. B., Sancar A., Little J. W., Rupp W. D. The uvrB gene of Escherichia coli has both lexA-repressed and lexA-independent promoters. Cell. 1982 Mar;28(3):523–530. doi: 10.1016/0092-8674(82)90207-0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Shimada K., Ogawa H., Tomizawa J. Studies on radiation-sensitive mutants of E. coli. II. Breakage and repair of ultraviolet irradiated intracellular DNA of phage lambda. Mol Gen Genet. 1968 May 3;101(3):245–256. doi: 10.1007/BF00271626. [DOI] [PubMed] [Google Scholar]
  26. Siegel E. C., Race H. M. Phenotypes of UV-sensitive uvrD3, recL152, and uvrE15 mutants of Escherichia coli. Mutat Res. 1981 Aug;83(1):49–59. doi: 10.1016/0027-5107(81)90070-1. [DOI] [PubMed] [Google Scholar]
  27. Van Sluis C. A., Mattern I. E., Paterson M. C. Properties of uvrE mutants of Escherichia coli K12. I. Effects of UV irradiation on DNA metabolism. Mutat Res. 1974 Dec;25(3):273–279. doi: 10.1016/0027-5107(74)90055-4. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES