Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells.

Abstract

Mutant lines of Chinese hamster ovary cells that show hypersensitivity to killing and mutagenesis by UV light were analyzed by genetic complementation analysis to determine whether defects in different gene loci might underlie a common cellular phenotype. To facilitate rapid screening of mutant clones, a procedure was devised that allowed presumptive complementation to be assessed on the basis of the frequency of UV-resistant cells after fusion by polyethylene glycol. Four classes were identified among 44 clones tested. By using drug-resistance markers for selection ofhybrid cells in crosses between UV mutant and wild type, a mutant from each of the four classes was shown to behave as phenotypically recessive. Hybrids were also isolated from crosses between each ofthe pair combinations ofthe four mutants. All such hybrids were relatively resistant to UV killing, providing confirmation of the complementation classes. When mutants representing the four UV-complementation classes were tested with the polyaromatic hydrocarbon 7-bromomethylbenz(a)anthracene, complementation was again seen for all pair combinations. These results suggest that each class ofmutants represents a biochemical defect that plays a common role in the repair of both UV-induced and chemically induced lesions in the DNA. The study ofmutant cell lines having defects in enzymatic pathways that repair damaged DNA is an important approach to understanding how cells recover from genetic injury and to elucidating the mechanisms of mutagenesis. Of the human mutations that are known to involve repair deficiencies, xeroderma pigmentosum (XP) has been the most intensively studied. XP fibroblasts and lymphoblasts are defective in the excision repair that operates on damage resulting from exposure to UV and many chemical mutagens and carcinogens (1, 2). The defects in XP appear to lie at or prior to the incision step (24) and may involve factors that play a role in enzymatic recognition ofdamaged DNA within chromatin (3, 4). Seven complementation classes of XP have been reported (5)(6)(7)(8).Recently, two techniques were introduced for isolating UVsensitive mutant clones from the Chinese hamster ovary (CHO) cell line (9, 10). Assay of DNA-repair replication indicated that these mutants, like XP mutants, have low or undetectable repair synthesis in response to UV (9, 10). These UV mutants of CHO cells, as well as alkylation-sensitive-mutants (9), provide an alternative to human mutant strains for studying the role of DNA repair in genetic damage. CHO cells can be readily manipulated in a quantitative manner, and they may allow isolation of mutants having repair deficiencies not represented among human mutants. In the.-present-study, we examined 44 UV mutants having pronounced sensitivity. We show here that they are genetically recessive and can be classified into four complementation groups on the basis of their responses to either UV or the chemical mutagen 7-bromomethylbenz(a)anthracene (7-BrMeBA). These results show that different genetic defects ...

DNA strand specificity for UV-induced mutations in mammalian cells.

“…which belongs to the second complementation group of UV-sensitive CHO mutants. This is the former first complementation group (22). At the UCLA meeting on DNA damage processing in Taos, N.M., in 1988, it was agreed that the nunibers of the first two complementation groups would be switched to allow correlation of the numbers of the cloned human DNA repair genes ERCC-1 and ERCC-2 with the numbers of their respective complementation groups (25).…”

Molecular cloning of the human nucleotide-excision-repair gene ERCC4.

“…Sci. USA 91 (1994) 2 series of genes that are required for the functioning of nucleotide-excision repair in mammalian cells (5,6). In a previous study, transfection of CHO UV41 cells with human DNA resulted in correction of cells to repair proficiency.…”

“…Seven human genes in this pathway have been identified and cloned by using mutants developed in rodent cells or derived from humans having the disorder xeroderma pigmentosum (XP) (4). Studies ofhamster and mouse mutant lines first identified five genetic complementation groups (5,6) having extreme UV-radiation sensitivity and excision-repair deficiency. From these groups of mutants, the complementing human genes ERCCI (7), XPD/ERCC2 (8), XPB/ERCC3 (9), and XPG/ERCCS (10) were cloned and shown to substantially overlap (9,(11)(12)(13) with the seven excision-deficient groups of XP (14).…”

“…These mutants and those in group 1, such as UV20 (5), are so far unique in their extreme sensitivities to both UV-radiation and cross-linking agents. These properties suggest that the complementing ERCCI and ERCC4 genes are involved in distinct but overlapping pathways of monoadduct and cross-link removal.…”

“…Since ERCC4 was assigned to chromosome 16 (23) (5) and its transformants were grown in either monolayer or suspension culture in minimum essential medium, a modification, supplemented with 10o (vol/vol) fetal bovine serum and antibiotics as reported (8). Colony-forming ability of transformants was determined after exposure to UV-radiation as reported (5). Triplicate 10-cm dishes were used for each dose point.…”