Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1986 Nov 11;14(21):8535–8556. doi: 10.1093/nar/14.21.8535

The purification of the Escherichia coli UvrABC incision system.

A T Yeung, W B Mattes, E Y Oh, G H Yoakum, L Grossman
PMCID: PMC311875  PMID: 3024108

Abstract

The UvrA, UvrB and UvrC proteins of Escherichia coli have been purified in good yields to homogeneity with rapid three- or four-step purification procedures. The cloned uvrA and uvrB genes were placed under control of the E. coli bacteriophage lambda PL promoter for amplification of expression. Expression of the uvrC gene could not be amplified by this strategy, however, subcloning of this gene into the replication-defective plasmid pRLM24 led to significant overproduction of the UvrC protein. The purified UvrA protein, with its associated ATPase activity, has a molecular weight of 114,000, the purified UvrB is an 84,000 molecular weight protein and the UvrC protein has a molecular weight of 67,000.

Full text

PDF
8541

Images in this article

Selected References

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

  1. 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]
  2. Caron P. R., Kushner S. R., Grossman L. Involvement of helicase II (uvrD gene product) and DNA polymerase I in excision mediated by the uvrABC protein complex. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4925–4929. doi: 10.1073/pnas.82.15.4925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. Sancar A., Clarke N. D., Griswold J., Kennedy W. J., Rupp W. D. Identification of the uvrB gene product. J Mol Biol. 1981 May 5;148(1):63–76. doi: 10.1016/0022-2836(81)90235-7. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. Yeung A. T., Mattes W. B., Oh E. Y., Grossman L. Enzymatic properties of purified Escherichia coli uvrABC proteins. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6157–6161. doi: 10.1073/pnas.80.20.6157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Yoakum G. H., Grossman L. Identification of E. coli uvrC protein. Nature. 1981 Jul 9;292(5819):171–173. doi: 10.1038/292171a0. [DOI] [PubMed] [Google Scholar]
  13. 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 Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES