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 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.     

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).     

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.     

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.     

Properties and applications of human DNA repair genes

The importance of understanding DNA repair processes is discussed in terms of the origins of human cancer. Several human repair genes have been mapped to specific human chromosomes using somatic cell hybrids. It is noteworthy that 3 of these genes lie in the same region of chromosome 19: genes ERCC1 and ERCC2, which are involved in nucleotide excision repair, and XRCC1, which is involved in the repair of strand breaks. The genes XRCC1 and ERCC2 were cloned from cosmid libraries prepared from DNA transformants of the CHO mutants EM9 and UV5, respectively. Analysis of the cDNA sequence of ERCC2 showed that the protein encoded by this gene is highly homologous (73%) to the RAD3 repair protein in the yeast Saccharomyces cerevisiae. Thus, the known properties of RAD3 combined with the high homology provide the first insight about the biochemical role of a human repair protein involved in the incision step of nucleotide excision repair. So far XRCC1 is the only cloned mammalian gene involved in repairing damage from ionizing radiation. The UV5 mutant line was also applied to problems in environmental mutagenesis by introducing the mouse cytochrome P(3)450 (P450IA2 subfamily) gene for metabolic activation of aromatic amines. We show in a rapid differential cytotoxicity assay with 2 compounds found in cooked beef (IQ, 2-amino-3-methylimidazo[4,5-f]quinoline and PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) that this gene is efficiently expressed in the transformed UV5P3 cells. Reversion of the repair deficiency in these cells will give a matched pair of cell lines that are metabolically proficient and repair deficient. Such lines will provide a rapid assay for genotoxic heterocyclic amines requiring activation.     

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.     

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.     

Evolution and mutagenesis of the mammalian excision repair gene ERCC-1.

The human DNA excision repair protein ERCC-1 exhibits homology to the yeast RAD10 repair protein and its longer C-terminus displays similarity to parts of the E. coli repair proteins uvrA and uvrC. To study the evolution of this 'mosaic' ERCC-1 gene we have isolated the mouse homologue. Mouse ERCC-1 harbors the same pattern of homology with RAD10 and has a comparable C-terminal extension as its human equivalent. Mutation studies show that the strongly conserved C-terminus is essential in contrast to the less conserved N-terminus which is even dispensible. The mouse ERCC-1 amino acid sequence is compatible with a previously postulated nuclear location signal and DNA-binding domain. The ERCC-1 promoter harbors a region which is highly conserved in mouse and man. Since the ERCC-1 promoter is devoid of all classical promoter elements this region may be responsible for the low constitutive level of expression in all mouse tissues and stages of embryogenesis examined.     

Evidence for involvement of HMGB1 protein in human DNA mismatch repair

Defects in human DNA mismatch repair predispose to cancer, but many components of the pathway have not been identified. We report here the identification and characterization of a novel component required for mismatch repair in human cells. A 30-kDa protein was purified to homogeneity by virtue of its ability to complement a depleted HeLa extract in repair of mismatched heteroduplexes. The complementing activity was identified as HMGB1 (the high mobility group box 1 protein), a non-histone chromatin protein that facilitates protein-protein interactions and recognizes DNA damage. Evidence is also presented that HMGB1 physically interacts with MutSalpha and is required at a step prior to the excision of mispaired nucleotide in mismatch repair.     

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.     

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.     

Isolation of human complexes proficient in nucleotide excision repair.

More than 20 polypeptides are required for the process of nucleotide excision repair (NER) in both human and yeast cells. This pathway of excision repair has most often been viewed as an ordered multi-step process involving steps of damage recognition, incision/excision and finally repair DNA synthesis. Here we present evidence for the existence of a complex of human NER proteins pre-assembled in the absence of damaged DNA. This multi-protein complex was initially isolated from HeLa cell extracts by affinity chromatography on a matrix containing the damage recognition protein XPA. Subsequent co-immunoprecipitation and gel filtration experiments demonstrated that a significant portion of the human NER proteins was present in the form of a high molecular weight complex and that these complexes, or repairosomes, were capable of performing all steps of NER in vitro . Consistent with studies indicating that DNA polymerasesdeltaandstraightepsiloncan both function in NER, these two polymerases are found in these repairosome complexes.     

XPA protein as a limiting factor for nucleotide excision repair and UV sensitivity in human cells

Nucleotide excision repair (NER) acts on a variety of DNA lesions, including damage induced by many chemotherapeutic drugs. Cancer therapy with such drugs might be improved by reducing the NER capacity of tumors. It is not known, however to what extent any individual NER protein is rate-limiting for any step of the repair reaction. We studied sensitivity to UV radiation and repair of DNA damage with regard to XPA, one of the core factors in the NER incision complex. About 150,000-200,000 molecules of XPA protein are present in NER proficient human cell lines, and no XPA protein in the XP-A cell line XP12RO. Transfected XP12RO cell lines expressing 50,000 or more XPA molecules/cell showed UV resistance similar to normal cells. Suppression of XPA protein to approximately 10,000 molecules/cell in a Tet-regulatable system modestly but significantly increased sensitivity to UV irradiation. No removal of cyclobutane pyrimidine dimers was detected in the SV40 immortalized cell lines tested. Repair proficient WI38-VA fibroblasts and transfected XP-A cells expressing 150,000 molecules of XPA/cell removed (6-4) photoproducts from the genome with a half-life of 1h. Cells in which XPA protein was reduced to about 10,000 molecules/cell removed (6-4) photoproducts more slowly, with a half-life of 3h. A reduced rate of repair of (6-4) photoproducts thus results in increased cellular sensitivity towards UV irradiation. These data indicate that XPA levels must be reduced to <10% of that present in a normal cell to render XPA a limiting factor for NER and consequent cellular sensitivity. To inhibit NER, it may be more effective to interfere with XPA protein function, rather than reducing XPA protein levels.     

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.     

Gamma-ray induced inhibition of DNA synthesis in ataxia telangiectasia fibroblasts is a function of excision repair capacity

The extent of the deficiency in γ-ray induced DNA repair synthesis in an ataxia telangiectasia (AT) human fibroblast strain was found to show no oxygen enhancement, consistent with a defect in the repair of base damage. Repair deficiency, but not repair proficiency, in AT cells were accompanied by a lack of inhibition of DNA synthesis (replicon initiation) neither γ-rays or the radiomimetic drug bleomycin. Experiments with 4-nitroquinoline 1-oxide indicated that lack of inhibition was specific for radiogenic type damage. Thus excision repair, perhaps by DNA strand incision or chromatin modification, appears to halt replicon initiation in irradiated repair proficient cells whereas in repair defective AT strains this putatively important biological function is inoperative.