PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
 
Mol Cell Biol. 1994 January; 14(1): 68–76.
PMCID: PMC358357

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

Abstract

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Barnes DE, Tomkinson AE, Lehmann AR, Webster AD, Lindahl T. Mutations in the DNA ligase I gene of an individual with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. Cell. 1992 May 1;69(3):495–503. [PubMed]
  • Bradford MM. 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. [PubMed]
  • Caldecott KW, Tucker JD, Thompson LH. Construction of human XRCC1 minigenes that fully correct the CHO DNA repair mutant EM9. Nucleic Acids Res. 1992 Sep 11;20(17):4575–4579. [PMC free article] [PubMed]
  • Chaganti RS, Schonberg S, German J. A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4508–4512. [PubMed]
  • Chan JY, Becker FF. Defective DNA ligase I in Bloom's syndrome cells. Simultaneous analysis using immunoblotting and the ligase-[32P]AMP adduct assay. J Biol Chem. 1988 Dec 5;263(34):18231–18235. [PubMed]
  • Chan JY, Becker FF, German J, Ray JH. Altered DNA ligase I activity in Bloom's syndrome cells. Nature. 1987 Jan 22;325(6102):357–359. [PubMed]
  • Chan JY, Thompson LH, Becker FF. DNA-ligase activities appear normal in the CHO mutant EM9. Mutat Res. 1984 May-Jun;131(5-6):209–214. [PubMed]
  • Dillehay LE, Thompson LH, Carrano AV. DNA-strand breaks associated with halogenated pyrimidine incorporation. Mutat Res. 1984 Mar-Apr;131(3-4):129–136. [PubMed]
  • Franke CA, Hruby DE. Expression and single-step purification of enzymatically active vaccinia virus thymidine kinase containing an engineered oligohistidine domain by immobilized metal affinity chromatography. Protein Expr Purif. 1993 Apr;4(2):101–109. [PubMed]
  • Ikejima M, Bohannon D, Gill DM, Thompson LH. Poly(ADP-ribose) metabolism appears normal in EM9, a mutagen-sensitive mutant of CHO cells. Mutat Res. 1984 Sep;128(2):213–220. [PubMed]
  • Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg HG. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8972–8976. [PubMed]
  • Jessberger R, Podust V, Hübscher U, Berg P. A mammalian protein complex that repairs double-strand breaks and deletions by recombination. J Biol Chem. 1993 Jul 15;268(20):15070–15079. [PubMed]
  • Krepinsky AB, Heddle JA, German J. Sensitivity of Bloom's syndrome lymphocytes to ethyl methanesulfonate. Hum Genet. 1979;50(2):151–156. [PubMed]
  • Kurihara T, Inoue M, Tatsumi K. Hypersensitivity of Bloom's syndrome fibroblasts to N-ethyl-N-nitrosourea. Mutat Res. 1987 Sep;184(2):147–151. [PubMed]
  • La Belle M, Linn S, Thompson LH. Apurinic/apyrimidinic endonuclease activities appear normal in the CHO-cell ethyl methanesulfonate-sensitive mutant, EM9. Mutat Res. 1984 Sep;141(1):41–44. [PubMed]
  • Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993 Apr 22;362(6422):709–715. [PubMed]
  • McDaniel LD, Schultz RA. Elevated sister chromatid exchange phenotype of Bloom syndrome cells is complemented by human chromosome 15. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7968–7972. [PubMed]
  • Minkler J, Stetka D, Jr, Carrano AV. An ultraviolet light source for consistent differential staining of sister chromatids. Stain Technol. 1978 Nov;53(6):359–360. [PubMed]
  • Perry P, Wolff S. New Giemsa method for the differential staining of sister chromatids. Nature. 1974 Sep 13;251(5471):156–158. [PubMed]
  • Ray JH, Louie E, German J. Different mutations are responsible for the elevated sister-chromatid exchange frequencies characteristic of Bloom's syndrome and hamster EM9 cells. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2368–2371. [PubMed]
  • Siciliano MJ, Carrano AV, Thompson LH. Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19. Mutat Res. 1986 Aug;174(4):303–308. [PubMed]
  • Söderhäll S, Lindahl T. Mammalian DNA ligases. Serological evidence for two separate enzymes. J Biol Chem. 1975 Nov 10;250(21):8438–8444. [PubMed]
  • Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Thompson LH, Brookman KW, Dillehay LE, Carrano AV, Mazrimas JA, Mooney CL, Minkler JL. A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange. Mutat Res. 1982 Aug;95(2-3):427–440. [PubMed]
  • Thompson LH, Brookman KW, Jones NJ, Allen SA, Carrano AV. Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange. Mol Cell Biol. 1990 Dec;10(12):6160–6171. [PMC free article] [PubMed]
  • Thompson LH, Brookman KW, Minkler JL, Fuscoe JC, Henning KA, Carrano AV. DNA-mediated transfer of a human DNA repair gene that controls sister chromatid exchange. Mol Cell Biol. 1985 Apr;5(4):881–884. [PMC free article] [PubMed]
  • Tomkinson AE, Lasko DD, Daly G, Lindahl T. Mammalian DNA ligases. Catalytic domain and size of DNA ligase I. J Biol Chem. 1990 Jul 25;265(21):12611–12617. [PubMed]
  • Tomkinson AE, Roberts E, Daly G, Totty NF, Lindahl T. Three distinct DNA ligases in mammalian cells. J Biol Chem. 1991 Nov 15;266(32):21728–21735. [PubMed]
  • Weiss B, Jacquemin-Sablon A, Live TR, Fareed GC, Richardson CC. Enzymatic breakage and joining of deoxyribonucleic acid. VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4. J Biol Chem. 1968 Sep 10;243(17):4543–4555. [PubMed]
  • Willis AE, Lindahl T. DNA ligase I deficiency in Bloom's syndrome. Nature. 1987 Jan 22;325(6102):355–357. [PubMed]
  • Willis AE, Weksberg R, Tomlinson S, Lindahl T. Structural alterations of DNA ligase I in Bloom syndrome. Proc Natl Acad Sci U S A. 1987 Nov;84(22):8016–8020. [PubMed]
  • Wilson IA, Niman HL, Houghten RA, Cherenson AR, Connolly ML, Lerner RA. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. [PubMed]
  • Yoo H, Li L, Sacks PG, Thompson LH, Becker FF, Chan JY. Alterations in expression and structure of the DNA repair gene XRCC1. Biochem Biophys Res Commun. 1992 Jul 31;186(2):900–910. [PubMed]
  • Zdzienicka MZ, van der Schans GP, Natarajan AT, Thompson LH, Neuteboom I, Simons JW. A Chinese hamster ovary cell mutant (EM-C11) with sensitivity to simple alkylating agents and a very high level of sister chromatid exchanges. Mutagenesis. 1992 Jul;7(4):265–269. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)