Molecular cloning of the human excision repair gene ERCC-6.
Molecular and Cellular Biology , Volume 10 p. 5806- 5813
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.
|*DNA Repair, *Genes, Structural, 0 (DNA Transposable Elements), 0 (DNA, Neoplasm), Animals, Blotting, Southern, Cell Line, Cell Survival/radiation effects, Cloning, Molecular, DNA Transposable Elements, DNA, Neoplasm/genetics, Hela Cells/metabolism, Human, Mutation, Restriction Mapping, Support, Non-U.S. Gov't, Transfection, Ultraviolet Rays|
|Molecular and Cellular Biology|
|Organisation||Erasmus MC: University Medical Center Rotterdam|
Troelstra, C, Odijk, H, de Wit, J, Westerveld, A, Thompson, L.H, Bootsma, D, & Hoeijmakers, J.H.J. (1990). Molecular cloning of the human excision repair gene ERCC-6. Molecular and Cellular Biology, 10, 5806–5813. Retrieved from http://hdl.handle.net/1765/3021