Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells

Keith W. Caldecott, James D. Tucker, Lawrence H. Stanker, Larry H. Thompson

Research output: Contribution to journalArticle

241 Scopus citations

Abstract

The human DNA repair protein XRCC1 was overexpressed as a histidine-tagged polypeptide (denoted XRCC1-His) inEscherichia coli and purified in milligram quantities by affinity chromatography. XRCC1-His complemented the mutant Chinese hamster ovary cell line EM9 when constitutively expressed from a plasmid or when introduced by electroporation. XRCC1-His directly interacted with human DNA ligase III in vitro to form a complex that was resistant to 2 M NaCI. XRCC1-His interacted equally well with DNA ligase III from Bloom syndrome, HeLa and MRC5 cells, indicating that Bloom syndrome DNA ligase III is normal in this respect. Detection of DNA ligase III on far Western blots by radiolabelled XRCC1-His indicated that the level of the DNA ligase polypeptide was reduced ∼4-fold in the mutant EM9 and also in EM-C11, a second member of the XRCC1 complementation group. Decreased levels of polypeptide thus account for most of the ∼6-fold reduced DNA ligase III activity observed previously in EM9. Immunodetection of XRCC1 on Western blots revealed that the level of this polypeptide was also decreased in EM9 and EM-C11 (>10-fold), indicating that the XRCC1-DNA ligase III complex is much reduced in the two CHO mutants.

Original languageEnglish (US)
Pages (from-to)4836-4843
Number of pages8
JournalNucleic Acids Research
Volume23
Issue number23
DOIs
StatePublished - 1995
Externally publishedYes

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)
  • Genetics

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