Altered DNA ligase III activity in the CHO EM9 mutant.
Delayed joining of DNA strand breaks and a high spontaneous level of sister-chromatid exchanges (SCEs) are characteristics of the mutant cell strain EM9 of Chinese hamster ovary (CHO) cells. The introduction of the human gene XRCC1 into EM9 cells reverts the phenotypic properties of EM9 to those of the wild type. We have investigated both DNA ligase activities and a protein which stimulates DNA ligase activity in mutant EM9 cells, XRCC1-transfectant H9T3-7-1 cells and wild-type AA8 cells. Our results, which demonstrate both a decreased DNA ligase activity in EM9 cells using poly(rA).oligo(dT) as substrate and a decreased ability of DNA ligase III to form a covalent DNA ligase III-adenylate intermediate with AMP, clearly indicate an altered DNA ligase III activity in the mutant. Furthermore, the AMP-binding capacity of DNA ligase III and its enzymatic activity with the synthetic polymer were restored after transfection of EM9 with the human XRCC1 gene. Immunoblotting data suggest that the XRCC1 gene does not code for DNA ligase III. In conclusion, the data indicate that the EM9 cell strain has an altered DNA ligase III activity that can be restored by the XRCC1 gene product.
Ljungquist
S
S
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Kenne
K
K
Olsson
L
L
Sandström
M
M
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D009154:Mutation; D000075223:Poly-ADP-Ribose Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D029867:Xenopus Proteins; D000249:Adenosine Monophosphate; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human