Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells.
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.
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, UK.
K W
KW
Caldecott
J D
JD
Tucker
L H
LH
Stanker
L H
LH
Thompson
D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D009154:Mutation; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D029867:Xenopus Proteins; D006639:Histidine; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human