Molecular analysis of ERCC2 mutations in the repair deficient hamster mutants UVL-1 and V-H1.
The cDNA sequence of the Chinese hamster ERCC2 nucleotide excision repair and transcription gene from the UVL-1 Chinese hamster ovary (CHO) mutant cell line and the V-H1 Chinese hamster V79 mutant line was analyzed. ERCC2 encodes a presumed ATP-dependent DNA helicase and is single copy in CHO lines due to the structural hemizygosity of chromosome 9. Both UVL-1 and V-H1 have intermediate levels of (6-4) photoproduct repair but are as highly UV sensitive as the group 2 mutants that have no detectable repair. Deficiency in cyclobutane dimer removal has also been shown for V-H1. In UVL-1, a single base substitution resulting in an Arg75-->Trp substitution in helicase domain Ia was identified. The equivalent amino acid position is also Arg in the human, mouse, Xiphophorus maculatus, Saccharomyces cerevisiae, and Schizosaccharomyces pombe homologs. In V-H1, a single base substitution resulting in a Thr46-->Ile substitution in helicase domain I (the ATP-binding domain) was identified in both alleles. The equivalent amino acid position is also Thr in the five homologs. Analysis of three V-H1 partial revertants revealed that they still have the original V-H1 mutation in both alleles, indicating that these are second site reversion events. Site-specific mutagenesis was used to introduce the Thr46-->Ile, Arg75-->Trp, and Lys48-->Arg (helicase domain I) mutations into a hamster ERCC2 expression plasmid. These plasmids each failed to confer UV resistance to group 2 mutant cells, further demonstrating that the changes identified are the causative mutations in V-H1 and UVL-1. Correlations between specific mutations, biochemical activities, and repair phenotype are discussed.
Kadkhodayan
S
S
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, CA 94551, USA. kadkhodayan2@llnl.gov
Salazar
E P
EP
Ramsey
M J
MJ
Takayama
K
K
Zdzienicka
M Z
MZ
Tucker
J D
JD
Weber
C A
CA
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004265:DNA Helicases; D004252:DNA Mutational Analysis; D004260:DNA Repair / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D018628:Gene Dosage; D017354:Point Mutation / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D051759:Xeroderma Pigmentosum Group D Protein
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011506:Proteins; D014157:Transcription Factors; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein