Genetic and biochemical characterization of the CHO-UV-1 mutant defective in postreplication recovery of DNA.
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.
Hentosh
P
P
Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado 80206.
Collins
A R
AR
Correll
L
L
Fornace
A J
AJ
Giaccia
A
A
Waldren
C A
CA
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000187:drug effects / Q000528:radiation effects*; D004249:DNA Damage; D004261:DNA Replication; D005020:Ethyl Methanesulfonate / Q000494:pharmacology*; D005260:Female; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007700:Kinetics; D008770:Methylnitrosourea / Q000494:pharmacology*; D009154:Mutation; D010053:Ovary; D014466:Ultraviolet Rays
D002110:Caffeine; D008770:Methylnitrosourea; D004247:DNA; D005020:Ethyl Methanesulfonate; D007041:Hypoxanthine Phosphoribosyltransferase