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Genetic and Biochemical Characterization of the CHO-UV-1 Mutant Defective in Postreplication Recovery of DNA¹

Eleanor Roosevelt Institute for Cancer Research [P. H., L. C., C. A. W.] Denver, Colorado 80206; Department of Radiology, University of Colorado Health Sciences Center [C. A. W.], Denver, Colorado 80262; University of Aberdeen, Department of Biochemistry, Marischal College, Aberdeen AB91AS, United Kingdom [A. R. S. C.]; Laboratory of Pathology, National Institutes of Health, Bethesda, Maryland 20892 [A. J. F.]; and Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 [A. G.]

The CHO-UV-1 mutant, a Chinese hamster ovary cell with defective postreplication recovery of DNA, is 2- to 4-fold more sensitive than its wild-type counterpart (CHO-77256) to the lethal effects of ethylating agents and UV radiation; it is also hypersensitive (10- to 20-fold) to some DNA-methylating and -cross-linking agents. We studied the CHO-UV-1 mutant further to define its phenotype in terms of DNA damage induction and repair, methyltransferase activity, and effects of caffeine on mutational and lethal responses. Both wild-type and CHO-UV-1 cells incurred similar levels and types of damage when exposed to UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, or N-methyl-N-nitrosourea. The rate and extent of repair of Micrococcus luteus endonuclease-sensitive sites after UV irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine were also equivalent in these two cell types. Twenty % of the initial endonuclease-sensitive sites induced in either cell line remained at 18 h after UV irradiation; approximately 8% of the sites after N-methyl-N'-nitro-N-nitrosoguanidine exposure were present in both parental and CHO-UV-1 cells after a 17-h repair period. Moreover, the ability of CHO-UV-1 to resynthesize and ligate DNA during excision repair was similar to that of its parent. Neither CHO-UV-1 nor CHO-77256 had appreciable levels of O⁶-methylguanine-DNA methyltransferase activity which ameliorates the cytotoxicity of alkylating agents. Caffeine, a known inhibitor of postreplication repair, decreased the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyltransferase locus by 40–55% in CHO-77256 but not in CHO-UV-1. These results rule out defective excision repair as a factor in the hypersensitivity of the CHO-UV-1 mutant to DNA-damaging agents. Hence, this cell line appears to derive from a mutation affecting nonexcision repair processes and should be useful in clarifying the mechanism(s) of postreplication recovery of DNA in mammalian cells.

¹ This work was supported by NIH Grants GM-34041, CA-36447, and HD-07197. This is contribution # 1018 from the Eleanor Roosevelt Institute.

² To whom requests for reprints should be addressed, at Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, P. O. Box 318, Memphis, TN 38101.

³ Present address: Department of Radiology and Radiation Biology, Colorado State University, Ft. Collins, CO 80523.

Received 9/26/89. Revised 12/27/89.

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