DNA-ligase activities appear in the CHO mutant EM9

Abstract

The Chinese hamster ovary (CHO) mutant strain EM9 was previously shown to be hypersensitive to killing by ethy methanesulfonate (EMS) and methyl methanesulfonate (MMS), to have a 12-fold increased baseline incidence of sister-chromatied exchanges (SCE), and to be defective in rejoining DNA and breaks after treatment with EMS, MMS, or X-rays. A study was performed to determine if the primary biochemical defect might be a DNA ligase. DNA-ligase activities were assayed and compared after separation of the multiple forms of ligase by AcA 34 gel-filtration chromatography of total cellular extracts. In EM9 cells the levels of the presumptive replicative forms, DNA ligase Ia (480 kd) and ligase Ib (240 kd) were about 50% amd 60%, respectively, of those in the parental AA8 cells, whereas DNA ligase II (80 kd) was unaltered in EM9. In a phenotypic revertant line (9RI) ligases Ia, IB, and II leldels were 35%, 37% and 100%, repsectively, of those in AA8. The reduced levels of ligases Ia and Ib in EM9 and 9RI cells are appraemtly not related directly op the mutant phenotype and may be attributable to the somewhat slower growth rates of these strains compared with those of AA8. To determine if the repair defect in EM9 might reside in the ability to induced DNA-ligase activity after treatment with a DNA-damaging agen, AA8 and EM9 cells were treated with MMS at 30 μg/ml for 60 min before preparing fractions for ligase assay. Under these conditions the activities of ligases Ia and Ib decreases 70–80% in both cell lines, but ligase II increased 2.0- and 2.6 -fold, respectively, in AA8 and EM9. As a further test of defective ligase activitues in EM9, assays were pergormed in the presence of 0.1 M NaCl or after heating the fractions for 10 min at 50μC. Although all 3 forms of ligase showed altered activity under both of these conditions, there were no significant differences between EMO and AA8 cells. These data combined with the above results provide strong evidence that the site of the primaru defect in EM9 is not either of the DNA ligases.