Molecular cloning of the human DNA excision repair gene ERCC-6

Journal Article · Thu Nov 01 04:00:00 EST 1990 · Molecular and Cellular Biology; (USA)

DOI:https://doi.org/10.1128/MCB.10.11.5806· OSTI ID:6271613

Troelstra, C; Odijk, H; de Wit, J; Westerveld, A; Thompson, L H; Bootsma, D; Hoeijmakers, J H ^[1]

---

1. Erasmus Univ. Rotterdam (Netherlands)

The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.

• OSTI ID: 6271613
• Journal Information: Molecular and Cellular Biology; (USA), Journal Name: Molecular and Cellular Biology; (USA) Vol. 10:11; ISSN MCEBD; ISSN 0270-7306
• Country of Publication: United States
• Language: English

Related Subjects

560120* -- Radiation Effects on Biochemicals
Cells
& Tissue Culture
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMALS
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CLONING
DNA REPAIR
ELECTROMAGNETIC RADIATION
EXCISION REPAIR
GENE AMPLIFICATION
GENES
HAMSTERS
HELA CELLS
MAMMALS
MAN
MUTANTS
PHENOTYPE
PRIMATES
PYRIMIDINE DIMERS
RADIATION EFFECTS
RADIATIONS
RADIOINDUCTION
RECOVERY
REPAIR
RODENTS
ULTRAVIOLET RADIATION
VERTEBRATES