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

Abstract	The human DNA repair protein XRCC1 was overexpressed as a histidine-tagged polypeptide (denoted XRCC1-His) in Escherichia 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 NaCl. 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 approximately 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 approximately 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.
Authors	K W Caldecott, J D Tucker, L H Stanker, L H Thompson
Journal	Nucleic acids research (Nucleic Acids Res) Vol. 23 Issue 23 Pg. 4836-43 (Dec 11 1995) ISSN: 0305-1048 [Print] England
PMID	8532526 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
Chemical References	Antibodies, Monoclonal DNA-Binding Proteins Poly-ADP-Ribose Binding Proteins Recombinant Proteins X-ray Repair Cross Complementing Protein 1 XRCC1 protein, human Xenopus Proteins Histidine DNA Ligases DNA Ligase ATP DNA ligase III alpha protein, Xenopus LIG3 protein, human
Topics	Animals Antibodies, Monoclonal CHO Cells (metabolism) Cricetinae DNA Ligase ATP DNA Ligases (metabolism) DNA Repair DNA-Binding Proteins (immunology, isolation & purification, metabolism, physiology) Escherichia coli (metabolism) HeLa Cells (metabolism) Histidine (metabolism) Humans Mutation Poly-ADP-Ribose Binding Proteins Recombinant Proteins (isolation & purification, metabolism) X-ray Repair Cross Complementing Protein 1 Xenopus Proteins

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