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

Keith W. Caldecott, Catherine K. McKeown, James D. Tucker, Siv Ljungquist, Larry H. Thompson

Research output: Contribution to journalArticle

400 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)68-76
Number of pages9
JournalMolecular and Cellular Biology
Volume14
Issue number1
StatePublished - 1994
Externally publishedYes

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Histidine
Cell Extracts
Affinity Chromatography
DNA Repair
Complementary DNA
DNA Ligases
Single-Stranded DNA Breaks
Sister Chromatid Exchange
Alkylating Agents
Cricetulus
Ionizing Radiation
Recombinant Proteins
DNA Ligase ATP
X-ray repair cross complementing protein 1
Ovary
Proteins
Phenotype
DNA
Genes

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Caldecott, K. W., McKeown, C. K., Tucker, J. D., Ljungquist, S., & Thompson, L. H. (1994). An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Molecular and Cellular Biology, 14(1), 68-76.

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. / Caldecott, Keith W.; McKeown, Catherine K.; Tucker, James D.; Ljungquist, Siv; Thompson, Larry H.

In: Molecular and Cellular Biology, Vol. 14, No. 1, 1994, p. 68-76.

Research output: Contribution to journalArticle

Caldecott, KW, McKeown, CK, Tucker, JD, Ljungquist, S & Thompson, LH 1994, 'An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III', Molecular and Cellular Biology, vol. 14, no. 1, pp. 68-76.
Caldecott KW, McKeown CK, Tucker JD, Ljungquist S, Thompson LH. An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Molecular and Cellular Biology. 1994;14(1):68-76.
Caldecott, Keith W. ; McKeown, Catherine K. ; Tucker, James D. ; Ljungquist, Siv ; Thompson, Larry H. / An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. In: Molecular and Cellular Biology. 1994 ; Vol. 14, No. 1. pp. 68-76.
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