Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants.
The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.
van Duin
M
M
Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
Janssen
J H
JH
de Wit
J
J
Hoeijmakers
J H
JH
Thompson
L H
LH
Bootsma
D
D
Westerveld
A
A
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003360:Cosmids; D006224:Cricetinae; D003412:Cricetulus; D004260:DNA Repair; D004261:DNA Replication / Q000528:radiation effects; D005796:Genes / Q000528:radiation effects; D005816:Genetic Complementation Test; D006801:Humans; D009154:Mutation; D014162:Transfection; D014466:Ultraviolet Rays