Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells.

Abstract

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 ceUs 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 aprt 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 alkaine 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.

XPD Lys751Gln and Asp312Asn Polymorphisms and Susceptibility to Skin Cancer: A Meta-Analysis of 17 Case-control Studies

“…It encodes an evolutionarily conserved ATPdependent DNA helicase protein, a basal transcription initiation factor complex TFIIH, which is essential for NER (Weber et al, 1988), basal transcription, and apoptosis (Wang et al, 1996). The XPD gene is involved in the DNA helix opening, and is capable of removing helix-distorting base lesions produced by ultraviolet light and chemical agents (Lehmann et al, 2001).…”

Molecular cloning of the human nucleotide-excision-repair gene ERCC4.

“…In a previous study, transfection of CHO UV41 cells with human DNA resulted in correction of cells to repair proficiency. Cloning of the gene was hampered by the apparent absence of repetitive sequences (22), which had been used to clone other genes such as ERCC2 (8). We used the sCos-1 chromosome 16-specific library for our initial transfection of UV41 because the ERCC4 gene had been assigned to this chromosome using CHO-human somatic cell hybrids (23).…”

“…The procedure used to clone ERCC4 is outlined in Fig. 1 (8). Cosmids were transfected into E. coli, and a selection for kanamycin resistance (conferred by the neo gene of sCos-1 present in the insert) was performed to rescue ERCC4 sequences (see Fig.…”

“…Studies ofhamster and mouse mutant lines first identified five genetic complementation groups (5,6) having extreme UV-radiation sensitivity and excision-repair deficiency. From these groups of mutants, the complementing human genes ERCCI (7), XPD/ERCC2 (8), XPB/ERCC3 (9), and XPG/ERCCS (10) were cloned and shown to substantially overlap (9,(11)(12)(13) with the seven excision-deficient groups of XP (14). The mutants in rodent complementation groups 6-11 have lesser degrees of UV sensitivity than groups 1-5 (15)(16)(17), and of the former set, only the gene correcting group 6, CSB/ERCC6, has been isolated (18).…”

“…Previous transfection studies using human genomic DNA resulted in corrected UV41 transformants without cloning ofthe ERCC4 gene (22). Since ERCC4 was assigned to chromosome 16 (23) (5) and its transformants were grown in either monolayer or suspension culture in minimum essential medium, a modification, supplemented with 10o (vol/vol) fetal bovine serum and antibiotics as reported (8). Colony-forming ability of transformants was determined after exposure to UV-radiation as reported (5).…”