ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3

Human ERCC2 genomic clones give efficient, stable correction of the nucleotide excision repair defect in UV5 Chinese hamster ovary cells. One clone having a breakpoint just 5' of classical promoter elements corrects only transiently, implicating further flanking sequences in stable gene expression. The nucleotide sequences of a cDNA clone and genomic flanking regions were determined. The ERCC2 translated amino acid sequence has 52% identity (73% homology) with the yeast nucleotide excision repair protein RAD3. RAD3 is essential for cell viability and encodes a protein that is a single-stranded DNA dependent ATPase and an ATP dependent helicase. The similarity of ERCC2 and RAD3 suggests a role for ERCC2 in both cell viability and DNA repair and provides the first insight into the biochemical function of a mammalian nucleotide excision repair gene.     

Isolation of the functional human excision repair gene ERCC5 by intercosmid recombination

The complete human nucleotide excision repair gene FRCC5 was isolated as a functional gene on overlapping cosmids. ERCC5 corrects the excision repair deficiency of Chinese hamster ovary cell line UV135, of complementation group 5. Cosmids that contained human sequences were obtained from a UV-resistant cell line derived from UV135 cells transformed with human genomic DNA. Individually, none of the cosmids complemented the UV135 repair defect; cosmid groups were formed to represent putative human genomic regions, and specific pairs of cosmids that effectively transformed UV135 cells to UV resistance were identified. Analysis of transformants derived from the active cosmid pairs showed that the functional 32-kbp ERCC5 gene was reconstructed by homologous intercosmid recombination. The cloned human sequences exhibited 100% concordance with the locus designated genetically as ERCC5 located on human chromosome 13q. Cosmid-transformed UV135 host cells repaired cytotoxic damage to levels about 70% of normal and repaired UV-irradiated shuttle vector DNA to levels about 82% of normal.     

Plant homologue of human excision repair gene ERCC1 points to conservation of DNA repair mechanisms

Nucleotide excision repair (NER), a highly versatile DNA repair mechanism, is capable of removing various types of DNA damage including those induced by UV radiation and chemical mutagens. NER has been well characterized in yeast and mammalian systems but its presence in plants has not been reported. Here it is reported that a plant gene isolated from male germline cells of lily (Lilium longiflorum) shows a striking amino acid sequence similarity to the DNA excision repair proteins human ERCC1 and yeast RAD10. Homologous genes are also shown to be present in a number of taxonomically diverse plant genera tested, suggesting that this gene may have a conserved function in plants. The protein encoded by this gene is able to correct significantly the sensitivity to the cross-linking agent mitomycin C in ERCC1-deficient Chinese hamster ovary (CHO) cells. These findings suggest that the NER mechanism is conserved in yeast, animals and higher plants.     

Activity of individual ERCC1 and XPF subunits in DNA nucleotide excision repair

ERCC1–XPF is a structure-specific nuclease with two subunits, ERCC1 and XPF. The enzyme cuts DNA at junctions where a single strand moves 5′ to 3′ away from a branch point with duplex DNA. This activity has a central role in nucleotide excision repair (NER), DNA cross-link repair and recombination. To dissect the activities of the nuclease it is necessary to investigate the subunits individually, as studies of the enzyme so far have only used the heterodimeric complex. We produced recombinant ERCC1 and XPF separately in Escherichia coli as soluble proteins. Activity was monitored by a sensitive dual incision assay for NER by complementation of cell extracts. XPF and ERCC1 are unstable in mammalian cells in the absence of their partners but we found, surprisingly, that ERCC1 alone could confer some repair to extracts from ERCC1-defective cells. A version of ERCC1 lacking the first 88 non-conserved amino acids was also functional. This indicated that a small amount of active XPF was present in ERCC1 extracts, and immunoassays showed this to be the case. Some repair in XPF-defective extracts could be achieved by adding ERCC1 and XPF proteins together, but not by adding only XPF. The results show for the first time that functional ERCC1–XPF can be formed from separately produced subunits. Protein sequence comparison revealed similarity between the ERCC1 family and the C-terminal region of the XPF family, including the regions of both proteins that are necessary for the ERCC1–XPF heterodimeric interaction. This suggests that the ERCC1 and XPF families are related via an ancient duplication.     

Complementation of the DNA repair-deficient swi10 mutant of fission yeast by the human ERCC1 gene.

In human cells DNA damage caused by UV light is mainly repaired by the nucleotide excision repair pathway. This mechanism involves dual incisions on both sides of the damage catalyzed by two nucleases. In mammalian cells XPG cleaves 3' of the DNA lesion while the ERCC1-XPF complex makes the 5' incision. The amino acid sequence of the human excision repair protein ERCC1 is homologous with the fission yeast Swi10 protein. In order to test whether these proteins are functional homologues, we overexpressed the human gene in a Schizosaccharomyces pombe swi10 mutant. A swi10 mutation has a pleiotropic effect: it reduces the frequency of mating type switching (a mitotic transposition event from a silent cassette into the expression site) and causes increased UV sensitivity. We found that the full-length ERCC1 gene only complements the transposition defect of the fission yeast mutant, while a C-terminal truncated ERCC1 protein also restores the DNA repair capacity of the yeast cells. Using the two-hybrid system of Saccharomyces cerevisiae we show that only the truncated human ERCC1 protein is able to interact with the S . pombe Rad16 protein, which is the fission yeast homologue of human XPF. This is the first example yet known that a human gene can correct a yeast mutation in nucleotide excision repair.     

The determination of relationship between “excision repair cross-complementing group 1” (ERCC1) gene T19007C and C8092A single nucleotide polymorphisms and clinicopathological parameters in non-small cell lung cancer

DNA repair plays a key role in prevention of carcinogenesis and one of the most important DNA repair mechanisms is nucleotide excision repair (NER) pathway. This pathway includes a number of genes such as excision repair cross-complementing group 1 (ERCC1) gene which are responsible for the 5′ incision of damaged DNA. A reduced DNA repair capacity associated with ERCC1 mRNA level has been observed in lung carcinogenesis. Two single nucleotide polymorphisms (SNPs) in ERCC1 gene, T19007C (rs11615) and C8092A (rs3212986), reportedly predict to affect the mRNA of ERCC1 in non-small cell lung cancer (NSCLC). To examine the role of two common SNPs in ERCC1 gene further, we conducted this study where 80 cases histopatologically diagnosed as NSCLC were genotyped. Genomic DNA was extracted from formalin-fixed, paraffin embedded tissues and two SNPs were analyzed using real-time PCR. The distributions of TT, TC, and CC genotypes of the T19007C SNP were 40, 44 and 16%, respectively. Significantly increased frequency of the patients carrying at least one 19007C allele was observed in early stage compared to advanced stage (P = 0.002). And also, the frequency of TC and CC genotypes significantly increased in younger patients compared to older patients (P = 0.035). Regarding C8092A SNP, the distribution of CC, CA, and AA genotypes was 38, 51 and 11%, respectively. There was no significant difference in the genotype distribution between C8092A SNP and clinicopathological parameters. This study indicated that harboring at least one 19007C allele may have protective effect in NSCLC.     

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.     

DNA repair variants,indoor tanning,and risk of melanoma

Although ultraviolet radiation (UV) exposure from indoor tanning has been linked to an increased risk of melanoma, the role of DNA repair genes in this process is unknown. We evaluated the association of 92 single nucleotide polymorphisms (SNPs) in 20 DNA repair genes with the risk of melanoma and indoor tanning among 929 patients with melanoma and 817 controls from the Minnesota Skin Health Study. Significant associations with melanoma risk were identified for SNPs in ERCC4, ERCC6, RFC1, XPC, MGMT, and FBRSL1 genes; with a cutoff of P < 0.05. ERCC6 and FBRSL1 gene variants and haplotypes interacted with indoor tanning. However, none of the 92 SNPs tested met the correction criteria for multiple comparisons. This study, based on an a priori interest in investigating the role of DNA repair capacity using variants in base excision and nucleotide excision repair, identified several genes that may play a role in resolving UV‐induced DNA damage.     

Studies on the Components of Mammalian Urine

Pectate lyase was purified approximately 29-fold to electrophoretic homogeneity from Pseudomonas marginalis N6301. A pectate lyase (PL; EC4.2.2.2) gene of the strain was cloned and expressed in Escherichiacoli. The nucleotides of the PL gene (pel) were sequenced. An open reading frame that encodes a polypeptide (molecular weight: 40,812) composed of 380 amino acids including a 29 amino acid signal peptide was assigned. The structural gene of pel consisted of 1140 base pairs. The nucleotide sequence of the 5′-flanking region of pel showed a consensus sequence of the promoter region of the pectin lyase gene (pnl) in P. marginalis N6301, a Pribnow box, and a ribosome binding site as found in E. coli.     

Differential processing of UV mimetic and interstrand crosslink damage by XPF cell extracts

We have recently developed a mammalian cell free assay in which interstrand crosslinks induce DNA synthesis in both damaged and undamaged plasmids co-incubated in the same extract. We have also shown using hamster mutants that both ERCC1 and XPF are required for the observed incorporation. Here, we show that extracts from an XPF patient cell line differentially process UV mimetic damage and interstrand crosslinks in vitro. XPF extracts are highly defective in the stimulation of repair synthesis by N-acetoxy-N- acetylaminofluorene, but are proficient in the stimulation of DNA synthesis by psoralen interstrand crosslinks. In addition, we show that extracts from the hamster UV140 mutant, which has high UV sensitivity, but moderate mitomycin C sensitivity, are similar in both assays to XPF cell extracts. These findings support the hypothesis that the activities of XPF in nucleotide excision repair (NER) and crosslink repair are separable, and that mutations in XPF patients result in the abolition of NER, but not recombinational repair pathways, which are likely to be essential as has been observed in ERCC1 homozygous –/– mice.     

Isolation of the functional human excision repair gene ERCC5 by intercosmid recombination

The complete human nucleotide exicision repair gene ERCC5 was isolated as a functional gene on overlapping cosmids. ERCC5 corrects the excision repair deficiency of Chinese hamster ovary cell line UV135, of complementation group 5. Cosmids that contained human sequences were obtained from a UV-resistant cell line derived from UV135 cells transformed with human genomic DNA. Individually, none of the cosmids complemented the UV135 repair defect; cosmid groups were formed to represent putative human genomic regions, and specific pairs of cosmids that effectively transformed UV135 cells to UV resistance were identified. Analysis of transformants derived from the active cosmid pairs showed that the functional 32-kbp ERCC5 gene was reconstructed by homologous intercosmid recombination. The cloned human sequences exhibited 100% concordance with the locus designated genetically as ERCC5 located on human chromosome 13q. Cosmid-transformed UV135 host cells repaired cytotoxic damage to levels about 70% of normal and repaired UV-irradiated shuttle vector DNA to levels about 82% of normal.     

Localization of the nucleotide excision repair gene ERCC6 to human chromosome 10q11-q21.

We have cloned the human DNA excision repair gene ERCC6 by virtue of its ability to correct the uv sensitivity of Chinese hamster overy cell mutant UV61. This mutant is a member of complementation group 6 of the nucleotide excision repair-deficient rodent mutants. By means of in situ hybridization and Southern blot analysis of mouse x human somatic cell hybrids, the gene was localized to human chromosome 10q11-q21. An RFLP detected within the ERCC6 locus can be helpful in linkage analysis.     

ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues.