title: The cloned human DNA excision repair gene ERCC-1 fails to correct xeroderma pigmentosum complementation groups A through I.
abstract: The human DNA excision repair gene ERCC-1 complements the ultraviolet light (UV) and mitomycin C (MMC) sensitivity of CHO mutants of complementation group 1. We have investigated whether ERCC-1 is the mutated gene in cell lines from xeroderma pigmentosum (XP) complementation groups A through I by analyzing the endogenous gene in XP cells and by introduction of the gene followed by repair assays. Our studies show that ERCC-1 is not deleted or grossly rearranged in representative cell lines of 9 XP groups. Furthermore, Northern blot analysis revealed correct transcription of ERCC-1 in all groups. The cloned human ERCC-1 gene was introduced into immortalized XP cells by DNA transfection (groups A, C, D, E and F). The presence of the integrated transfected sequences was verified on Southern blots and by selection for 2 dominant marker genes that flank the ERCC-1 gene on the transfected cos43-34 DNA. ERCC-1 failed to confer a normal UV survival and UV-induced unscheduled DNA synthesis (UDS) to transfected populations. In the case of the remaining XP complementation groups (B, G, H and I), nuclear microinjection was used to introduce an ERCC-1 cDNA construct driven by an SV40 promoter into primary fibroblasts. Coinjection of the SV40 large T gene and analysis of its expression served as a control for the injection. The ERCC-1 cDNA failed to induce increased levels of UDS in the microinjected fibroblasts. We infer from these experiments that ERCC-1 is not the mutated gene in the 9 XP complementation groups examined. From a similar type of experiments we conclude that ERCC-1 is not the defective gene in UV-sensitive Cockayne's syndrome cells.
authors.affiliation: Department of Cell Biology and Genetics, Erasmus University Rotterdam, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: van Duin
authors.forename: G
authors.initials: G
authors.lastname: Vredeveldt
authors.forename: L V
authors.initials: LV
authors.lastname: Mayne
authors.forename: H
authors.initials: H
authors.lastname: Odijk
authors.forename: W
authors.initials: W
authors.lastname: Vermeulen
authors.forename: B
authors.initials: B
authors.lastname: Klein
authors.forename: G
authors.initials: G
authors.lastname: Weeda
authors.forename: J H
authors.initials: JH
authors.lastname: Hoeijmakers
authors.forename: D
authors.initials: D
authors.lastname: Bootsma
authors.forename: A
authors.initials: A
authors.lastname: Westerveld
mesh_terms: D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004260:DNA Repair; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D006801:Humans; D008845:Microinjections; D014162:Transfection; D014983:Xeroderma Pigmentosum / Q000235:genetics*
chemical_list: D004247:DNA