Role of the human ERCC-1 gene in gene-specific repair of cisplatin-induced DNA damage.

The human excision repair gene ERCC-1 gene restores normal resistance to UV and mitomycin C in excision repair deficient chinese hamster ovary cells of complementation group 1. To investigate the involvement of the ERCC-1 gene in gene-specific repair of bulky lesions, we have studied the removal of damage induced by the antitumor agent cisplatin in CHO mutant 43-3B cells of group 1, with or without transfection with the ERCC-1 gene. Firstly, we determined the contribution of the ERCC-1 gene to the repair of interstrand crosslinks (ICL) induced by cisplatin and found efficient removal of ICLs from the dihydrofolate reductase (DHFR) gene in the ERCC-1 transfected 43-3B cells. We then assessed the contribution of ERCC-1 to the repair of intrastrand adducts (IA) induced by cisplatin. Compared to the wild-type parental cell line, the ERCC-1 transfected 43-3B cells repaired the IAs in the DHFR gene inefficiently. Thus, our data suggest that the ERCC-1 gene is more involved in the repair of interstrand crosslinks than in the removal of intrastrand adducts.     

Molecular cloning and biological characterization of the human excision repair gene ERCC-3.

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion (L. H. Thompson, A. V. Carrano, K. Sato, E. P. Salazar, B. F. White, S. A. Stewart, J. L. Minkler, and M. J. Siciliano, Somat. Cell. Mol. Genet. 13:539-551, 1987).     

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

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.     

Gene amplification in Chinese hamster DNA repair deficient mutants

In order to study the possible relationship between gene amplification and DNA repair we analyzed the amplification of the CAD gene in four mutants hypersensitive to UV light (CHO43RO, CHO7PV, UV5 and UV61) isolated in vitro from Chinese hamster cell lines (CHO-K1 and AA8). These mutants are characterized by different defects in the nucleotide excision repair mechanism and represent complementation groups 1, 9, 2, and 6 respectively. To evaluate the amplification ability of each cell line we measured the rate of appearance of PALA resistant clones with the Luria and Delbrück fluctuation test. Resistance to PALA is mainly due to amplification of the CAD gene. In the mutants CHO43RO, UV5 and CHO7PV we reproducibly found an amplification rate lower than in the parental cell lines (2–5 times), while in UV61 the amplification rate was about 4 times higher. This result indicates that each mutant is characterized by a specific amplification ability and that the unefficient removal of UV induced DNA damage can be associated with either a higher or a lower amplification rate. However, the analysis of randomly isolated CHO-K1 clones with normal UV sensitivity has shown variability in their amplification ability, making it difficult to relate the specific amplification ability of the mutants to the DNA repair defect and suggesting clonal heterogeneity of the parental population.     

Cytogenetical characterisation of Chinese hamster 43-3B transferants with the amplified or non-amplified human DNA repair gene ERCC-1

A comparative study on the biological responses to different mutagens (UV, 4NQO, MMC, MMS and EMS) was made on CHO wild-type cells (CHO-9), its UV-hypersensitive mutant 43-3B, and 2 types of its transferants, i.e., one containing a few copies of the human repair gene ERCC-1 and the other having more than 100 copies of ERCC-1 (due to gene amplification). Cell survival, chromosomal aberrations and SCEs were used as biological end-points. The spontaneous frequency of chromosomal aberrations in the transferants was less than found in 43-3B mutant cells, but still 2-3 times higher than in wild-type CHO cells. The spontaneous frequency of SCEs in the transferants was less than in 43-3B and similar to that of wild-type cells. The induction of SCEs by all tested agents in transferants was similar to that found in CHO-9 cells, while the mutant is known to respond with higher frequencies. ERCC-1 also bestowed resistance to MMS and EMS on the mutant to induction of chromosomal aberrations and cell killing to levels comparable with those of the wild-type strain. On the other hand ERCC-1 could not completely regain the repair proficiency against cell killing and induction of chromosomal aberrations by UV or MMC to the wild-type level. These results suggest that the ERCC-1 corrects the repair defect in CHO mutant cells, but it is unable to rectify fully the defect; probable reasons for this are discussed. However, amplified transferants (having more than 100 copies of the ERCC-1 gene) restored the impaired repair function in 43-3B to UV-, MMC- or 4NQO-induced DNA damage better than non-amplified transferants with a few copies of the ERCC-1. This difference may be due to the high amount of gene product involved in the excision repair process in the amplified cells.     

Human repair gene restores normal pattern of preferential DNA repair in repair defective CHO cells.

The pattern of preferential DNA repair of UV-induced pyrimidine dimers was studied in repair-deficient Chinese hamster ovary (CHO) cells transfected with the human excision repair gene, ERCC-1. Repair efficiency was measured in the active dihydrofolate reductase (DHFR) gene and in its flanking, non-transcribed sequences in three cell lines: Wild type CHO cells, a UV-sensitive excision deficient CHO mutant, and the transfected line of the mutant carrying the expressed ERCC-1 gene. The CHO cells transformed with the human ERCC-1 gene repaired the active DHFR gene much more efficiently than the non-transcribed sequences, a pattern similar to that seen in wild type CHO cells. This pattern differs from that previously reported in CHO cells transfected with the denV gene of bacteriophage T4, in which both active and non-transcribed DNA sequences were efficiently repaired (Bohr and Hanawalt, Carcinogenesis 8: 1333-1336, 1987). The ERCC-1 gene product may specifically substitute for the repair enzyme present in normal hamster cells while the denV product, T4 endonuclease V, does not be appear to be constrained in its access to inactive chromatin.     

Requirement for ERCC-1 and ERCC-3 gene products in DNA excision repair in vitro. Complementation using rodent and human cell extracts.

Numerous rodent cell lines exist that have defects in nucleotide excision repair of DNA caused by alterations in genes that fall into 10 different complementation groups. The precise roles in the repair of these genes are unknown. We report here that extracts from Chinese hamster ovary cells of excision repair-defective complementation groups 1 and 3 are defective in DNA excision repair in a cell-free system. In vitro complementation can be achieved by mixing extracts from the two groups with one another. In addition, extracts from a human cell line representing xeroderma pigmentosum complementation group B could complement rodent complementation group 1 extracts, but not group 3 extracts. This is consistent with an identity of the ERCC-3 and xeroderma pigmentosum group B genes. Cellular evidence points toward a defect in the incision of damaged DNA in group 1 and 3 mutants. Since the ERCC-1 and ERCC-3 proteins are required for the in vitro reaction, it appears that both gene products are directly involved in the enzymatic incision of damaged DNA, or in preincision reactions. The experiments reported here provide the biochemical basis of an approach to analyze the function of these nucleotide excision repair proteins.     

Differential repair and replication of damaged DNA in ribosomal RNA genes in different CHO cell lines

We studied the repair of psoralen adducts in the pol I-transcribed ribosomal RNA (rRNA) genes of excision repair competent Chinese hamster ovary (CHO) cell lines, their UV sensitive mutant derivatives, and their UV resistant transformants, which express a human excision repair gene. In the parental cell line CHO-AA8, both monoadducts and interstrand crosslinks are removed efficiently from the rRNA genes, whereas neither adduct is removed in the UV sensitive derivative UV5; removal of both adducts is restored in the UV resistant transformant CHO-5T4 carrying the human excision repair gene ERCC-2. In contrast, removal of psoralen adducts from the rRNA genes is not detected in another parental CHO cell line CHO-9, neither in its UV sensitive derivative 43-3B, nor in its UV resistant transformant 83-G5 carrying the human excision repair gene ERCC-1. In contrast to such intergenomic heterogeneity of repair, persistence of psoralen monoadducts during replication of the rRNA genes occurs equally well in all CHO cell lines tested. From these data, we conclude that: 1) the repair efficiency of DNA damage in the rRNA genes varies between established parental CHO cell lines; 2) the repair pathways of intrastrand adducts and interstrand crosslinks in mammalian cells share, at least, one gene product, i.e., the excision repair gene ERCC-2; 3) replicational bypass of psoralen monoadducts at the CHO rRNA locus occurs similarly on both DNA strands.     

A presumed DNA helicase encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's syndrome

The human gene ERCC-3 specifically corrects the defect in an early step of the DNA excision repair pathway of UV-sensitive rodent mutants of complementation group 3. The predicted 782 amino acid ERCC-3 protein harbors putative nucleotide, chromatin, and helix-turn-helix DNA binding domains and seven consecutive motifs conserved between two superfamilies of DNA and RNA helicases, strongly suggesting that it is a DNA repair helicase. ERCC-3-deficient rodent mutants phenotypically resemble the human repair syndrome xeroderma pigmentosum (XP). ERCC-3 specifically corrects the excision defect in one of the eight XP complementation groups, XP-B. The sole XP-B patient presents an exceptional conjunction of two rare repair disorders: XP and Cockayne's syndrome. This patient's DNA contains a C----A transversion in the splice acceptor sequence of the last intron of the only ERCC-3 allele that is detectably expressed, leading to a 4 bp insertion in the mRNA and an inactivating frameshift in the C-terminus of the protein. Because XP is associated with predisposition to skin cancer, ERCC-3 can be considered a tumor-preventing gene.     

Structure and expression of the human XPBC/ERCC-3 gene involved in DNA repair disorders xeroderma pigmentosum and Cockayne's syndrome

The human XPBC/ERCC-3 was cloned by virtue of its ability to correct the excision repair defect of UV-sensitive rodent mutants of complementation group 3. The gene appeared to be in addition implicated in the human, cancer prone repair disorder xeroderma pigmentosum group B, which is also associated with Cockayne's syndrome. Here we present the genomic architecture of the gene and its expression. The XPBC/ERCC-3 gene consists of at least 14 exons spread over approximately 45 kb. Notably, the donor splice site of the third exon contains a GC instead of the canonical GT dinucleotide. The promoter region, first exon and intron comprise a CpG island with several putative GC boxes. The promoter was confined to a region of 260 bp upstream of the presumed cap site and acts bidirectionally. Like the promoter of another excision repair gene, ERCC-1, it lacks classical promoter elements such as CAAT and TATA boxes, but it shares with ERCC-1 a hitherto unknown 12 nucleotide sequence element, preceding a polypyrimidine track. Despite the presence of (AU)-rich elements in the 3'-untranslated region, which are thought to be associated with short mRNA half-life actinomycin-D experiments indicate that the mRNA is very stable (t 1/2 greater than 3h). Southern blot analysis revealed the presence of XPBC/ERCC-3 cross-hybridizing fragments elsewhere in the genome, which may belong to a related gene.     

Genetic complementation between UV-sensitive CHO mutants and xeroderma pigmentosum fibroblasts

The purpose of this study was to determine the feasibility of doing complementation analysis between DNA-repair mutants of CHO cells and human fibroblasts based on the recovery of hybrid cells resistant to DNA damage. Two UV-sensitive CHO mutant lines, UV20 and UV41, which belong to different genetic complementation groups, were fused with fibroblasts of xeroderma pigmentosum in various complementation groups. Selection for complementing hybrids was performed using a combination of ouabain to kill the XP cells and mitomycin C to kill the CHO mutants. Because the frequency of viable hybrid clones was generally < 10⁻⁶ and the frequency of revertants of each CHO mutant was 2×10⁻⁷, putative hybrids required verification. The hybrid character of clones was established by testing for the presence of human DNA in a dot-blot procedure.

Hybrid clones were obtained from 9 of the 10 different crosses involving 5 complementation groups of XP cells. The 4 attempted crosses with 2 other XP groups yielded no hybrid colonies. Thus, a definitive complementation analysis was not possible. Hybrids were evaluated for their UV resistance using a rapid assay that measures differential cytotoxicity (DC). All 9 hybrids were more resistant than the parental mutant CHO and XP cells, indicating that in each case complementation of the CHO repair defect by a human gene had occurred. 3 hybrids were analyzed for their UV-radiation survival curves and shown to be much more resistant that the CHO mutants but less resistant than normal CHO cells. With 2 of these hybrids, sensitive subclones, which had presumably lost the complementing gene, were found to have similar sensitivity to the parental CHO mutants. We conclude that the extremely low frequency of viable hybrids in this system limits the usefulness of the approach. The possibility remains that each of the nonhybridizing XP strains could be altered in the same locus as one of the CHO mutants.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Localization of the xeroderma pigmentosum group B-correcting gene ERCC3 to human chromosome 2q21

The human excision-repair gene ERCC3 was cloned after DNA-mediated gene transfer to the uv-sensitive Chinese hamster ovary mutant cell line 27-1, a member of complementation group 3 of the excision-defective rodent cell lines. The ERCC3 gene specifically corrects the DNA repair defect of xeroderma pigmentosum (XP) complementation group B, which displays the clinical symptoms of XP as well as of another rare excision-repair disorder, Cockayne syndrome. The gene encodes a presumed DNA and chromatin binding helicase, involved in early steps of the excision-repair pathway. ERCC3 was previously assigned to human chromosome 2 (L.H. Thompson, A.V. Carrano, K. Sato, E.P. Salazar, B.F. White, S.A. Stewart, J.L. Minkler, and M.J. Siciliano (1987) Somat. Cell Genet. 13: 539-551). Here we report its subchromosomal localization in the q21 region of chromosome 2 via somatic cell hybrids containing a translocated chromosome 2 and in situ hybridization with fluorescently labeled ERCC3 probes.     

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 in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

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.     

Genetic analysis of X-ray-sensitive mutants of the CHO cell line

The genetic diversity of 6 X-ray-sensitive (xrs) mutants of the CHO cell line has been investigated. Hybrids were constructed by fusing ouabain- and 6-thioguanine-resistant cells to ouabain- and 6-thioguanine-sensitive cells and selecting in HAT and ouabain medium. Hybrids were examined for ploidy and X-ray sensitivity. Crosses between xrs mutants and wild-type showed that each mutant was recessive. Crosses between different xrs mutants showed that all were in the same complementation group. Although all the mutants are primarily sensitive to ionizing radiation and bleomycin, and all have a defect in double-strand break rejoining, their cross-sensitivity to other DNA-damaging agents differed to some degree. One explanation is that this repair gene is involved in a pleiotropic response to DNA damage.     

Intermediate (6-4) photoproduct repair in Chinese hamster V79 mutant V-H1 correlates with intermediate levels of DNA incision and repair replication

A DNA-repair mutant isolated from Chinese hamster V79 cells, V-H1, has been characterized as having only slightly reduced unscheduled DNA synthesis (UDS) and intermediate levels of DNA incision and repair replication after UV exposure. This observation was unexpected, since V-H1 has been shown by genetic complementation analysis to belong to the UV5 complementation class (i.e., class 2), exhibiting equivalent UV hypersensitivity and hypermutability as UV5 cells, which are defective in incision, UDS and repair replication. We have examined the repair of cyclobutane dimers and (6-4) photoproducts in V-H1 and V79 cells and shown that V-H1 cells are deficient in cyclobutane dimer repair, but exhibit intermediate (6-4) photoproduct repair, unlike UV5 cells which are completely deficient in (6-4) photoproduct repair. Our results confirm observations made in other UV-hypersensitive Chinese hamster cell mutants in CHO complementation class 2, and suggest that the gene affected in these mutants (ERCC2) may be involved in at least two distinct repair pathways in hamster cells.