Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells.
The UV-sensitive Chinese hamster ovary (CHO) cell line UV5, which is defective in the incision step of nucleotide excision repair, was used to identify and clone a complementing human gene, ERCC2, and to study the repair process. Genomic DNA from a human-hamster hybrid cell line was sheared and cotransferred with pSV2gpt plasmid DNA into UV5 cells to obtain five primary transformants. Transfer of sheared DNA from one primary transformant resulted in a secondary transformant expressing both gpt and ERCC2. The human repair gene was identified with a probe for Alu-family repetitive sequences. For most primary, secondary, and cosmid transformants, survival after UV exposure showed a return to wild-type levels of resistance. The levels of UV-induced mutation at the aprt locus for secondary and cosmid transformants varied from 50 to 130% of the wild-type level. Measurements of the initial rate of UV-induced strand incision by alkaline elution indicated that, whereas the UV5 rate was 3% of the wild-type level, rates of cosmid-transformed lines were similar to that of the wild type, and the secondary transformant rate was about 165% of the wild-type rate. Analysis of overlapping cosmids determined that ERCC2 is between 15.5 and 20 kilobases and identified a closely linked gpt gene. Cosmids were obtained with functional copies of both ERCC2 and gpt. ERCC2 corrects only the first of the five CHO complementation groups of incision-defective mutants.
Weber
C A
CA
Biomedical Sciences Division, Lawrence Livermore National Laboratory, California 94550.
Salazar
E P
EP
Stewart
S A
SA
Thompson
L H
LH
D000818:Animals; D002460:Cell Line; D002888:Chromosomes, Human, Pair 19; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA / Q000235:genetics*; D004260:DNA Repair; D004262:DNA Restriction Enzymes; D005796:Genes; D006801:Humans; D009154:Mutation; D009693:Nucleic Acid Hybridization; D014170:Transformation, Genetic; D014960:X Chromosome
D004247:DNA; D004262:DNA Restriction Enzymes