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 part 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.
- Research Organization:
- Biomedical Sciences Div., Lawrence Livermore National Lab., P.O. Box 5507, Livermore, CA (US)
- DOE Contract Number:
- W-7405-ENG-48
- OSTI ID:
- 5131564
- Journal Information:
- Mol. Cell. Biol.; (United States), Journal Name: Mol. Cell. Biol.; (United States) Vol. 8:3; ISSN MCEBD
- Country of Publication:
- United States
- Language:
- English
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560120* -- Radiation Effects on Biochemicals
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59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMAL CELLS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CELL CONSTITUENTS
CHO CELLS
CLONING
DEFECTS
DNA
DNA REPAIR
DNA REPLICATION
DNA-CLONING
ELECTROMAGNETIC RADIATION
EXCISION REPAIR
GENE RECOMBINATION PROTEINS
GENETIC MAPPING
HYBRIDIZATION
KINETICS
MAMMALS
MAN
MAPPING
MOLECULAR BIOLOGY
MUTAGENESIS
MUTANTS
MUTATIONS
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
NUCLEOPROTEINS
ORGANIC COMPOUNDS
PLASMIDS
PRIMATES
PROTEINS
RADIATION INDUCED MUTANTS
RADIATIONS
RADIOINDUCTION
REACTION KINETICS
RECOMBINANT DNA
RECOVERY
REPAIR
STRAND BREAKS
ULTRAVIOLET RADIATION
VERTEBRATES