Christine A. Weber

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.

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

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 cells 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 alkaline 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.

Refined mapping of the three DNA repair genes, ERCC1, ERCC2, and XRCC1, on human chromosome 19.

Three DNA repair genes, ERCC1, ERCC2, and XRCC1, have been regionally mapped on human chromosome 19. ERCC2 and XRCC1 have been assigned to bands q13.2----q13.3 by in situ hybridization using fluorescently-labeled cosmid probes. ERCC1 and ERCC2 have been found to be separated by less than 250 kb by large fragment restriction enzyme site mapping.

Recent Progress with the DNA Repair Mutants of Chinese Hamster Ovary Cells

SUMMARY Repair-deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant, which is noted for its very high sister-chromatid exchange frequencies. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to chlorodeoxyuridine (CldUrd). After EcoRI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25–30 kilobases (kb) was present. Since the DNA molecules used to produce these transformants were sheared to <50kb in size, the correcting gene should be small enough to clone in a cosmid vector. Using drug-resistance markers to select for hybrids after fusion, we have done complementation experiments with ultraviolet light (u.v.)-sensitive mutants and have identified a sixth complementation group, line UV61. Additionally, CHO mutants UV27-1 and MMC-2, isolated in other laboratories, were found to belong to UV group 3, which is represented by line UV24. To study the behaviour of transfected DNA molecules in repair-deficient cells, we treated plasmid pSV2gpt with either u.v. radiation or cis-diamminedichloroplatinum(II) (cis-DDP) and introduced the damaged DNA into normal CHO cells (AA8) and mutants UV4 and UV5. Unrepaired damage to the plasmid was indicated by loss of colony-forming ability of the transfected cells in selective medium containing mycophenolic acid. With u.v. damage, the differential survival of the cell lines was similar to that seen when whole cells are treated with u.v. However, with cis-DDP damage, mutant UV4 did not exhibit the extreme hypersensitivity (50-fold) that occurs when cells are treated. This result suggests that UV4 cells may be able to repair cross-links in transfected DNA.

Complementation of repair gene mutations on the hemizygous chromosome 9 in CHO: a third repair gene on human chromosome 19.

A human DNA repair gene, ERCC2 (Excision Repair Cross Complementing 2), was assigned to human chromosome 19 using hybrid clone panels in two different procedures. One set of cell hybrids was constructed by selecting for functional complementation of the DNA repair defect in mutant CHO UV5 after fusion with human lymphocytes. In the second analysis, DNAs from an independent hybrid panel were digested with restriction enzymes and analyzed by Southern blot hybridization using DNA probes for the three DNA repair genes that are located on human chromosome 19: ERCC1, ERCC2, and X-Ray Repair Cross Complementing 1 (XRCC1). The results from hybrids retaining different portions of this chromosome showed that ERCC2 is distal to XRCC1 and in the same region of the chromosome 19 long arm (q13.2-q13.3) as ERCC1, but on different MluI macrorestriction fragments. Similar experiments using a hybrid clone panel containing segregating Chinese hamster chromosomes revealed the hamster homologs of the three repair genes to be part of a highly conserved linkage group on Chinese hamster chromosome number 9. The known hemizygosity of hamster chromosome 9 in CHO cells can account for the high frequency at which genetically recessive mutations are recovered in these three genes in CHO cells. Thus, the conservation of linkage of the repair genes explains the seemingly disproportionate number of repair genes identified on human chromosome 19.

Expression of excision repair genes in non-malignant bone marrow from cancer patients

The patterns of expression of 3 human DNA-repair genes (ERCC1, ERCC2, ERCC6) were assessed in 52 bone-marrow specimens obtained from cancer patients prepared for autologous bone-marrow transplantation. Marrow was collected prior to the initiation of treatment in patients with sarcoma or testicular cancer; marrow was collected after initial cytoreductive therapy for patients with non-Hodgkins lymphoma, Hodgkins disease, and other tumors. Slot-blot analysis of marrow RNA showed a bimodal pattern of ERCC1, ERCC2 and ERCC6 gene expression with relative expression values ranging more than 200-fold. This pattern was seen in all patient groups and appeared to be independent of whether or not patients had received prior chemotherapy. In all patient groups, when expression was low for ERCC1, expression was also low for ERCC2 and ERCC6, suggesting that expression of these genes may be coordinated within an individual although they are located on two different chromosomes. Southern blot analyses of Pst I digests of DNA from 6 bone-marrow samples indicate no differences in ERCC1 gene copy number between high expressors and low expressors. There is absence of restriction fragment length polymorphism for ERCC1 suggesting that the different levels of expression in high and low expressors were not due to major deletions or rearrangements of the ERCC1 gene. We conclude that expression of these ERCC genes may vary widely between individuals, and that within an individual, their expression may be linked and coordinated by a common regulatory mechanism.

DNA repair characteristics and mutations in the ERCC2 DNA repair and transcription gene in a trichothiodystrophy patient

Patient TTD183ME is male and has typical trichothiodystrophy characteristics, including brittle hair, ichthyosis, characteristic face with receding chin and protruding ears, sun sensitivity, and mental and growth retardation. The relative amount of NER carried out by a TTD183ME fibroblast cell strain after ultraviolet (UV) exposure was ∼65% of normal as determined by a method that converts repair patches into quantifiable DNA breaks. UV survival curves show a reduction in survival only at doses greater than 4 J/m2. Nucleotide sequence analysis of the ERCC2 (XPD) DNA repair and transcription gene cDNA revealed both a Leu461‐to‐Val substitution and a deletion of amino acids 716–730 in one allele and an Ala725‐to‐Pro substitution in the other allele. The first allele has also been reported in one xeroderma pigmentosum group D patient and two other trichothiodystrophy patients, while the second allele has not been previously reported. Comparisons suggest that the mutation of Ala725 to Pro correlates with TTD with intermediate UV sensitivity. Hum Mutat 9:519–525, 1997.

Cyclobutane-pyrimidine dimer excision in UV-sensitive CHO mutants and the effect of the human ERCC2 repair gene

Using a radiochromatographic assay, we have examined cis-syn cyclobutane-pyrimidine dimer removal after ultraviolet irradiation in cell lines representative of the first 6 complementation groups of Chinese hamster ovary DNA nucleotide excision repair mutants. AA8, the CHO cell line from which these mutants were derived, consistently showed normal dimer excision for a rodent cell. The mutants uniformly exhibited no significant dimer excision within the limits of determination. Additionally, V-H1, a mutant belonging to complementation group 2 and derived from V79 hamster cells, exhibited no dimer excision. Two UV5 derived transformants that carry the complementing human ERCC2 repair gene showed a capacity for dimer excision comparable to the AA8 wild-type cells.

Molecular analysis of CXPD mutations in the repair-deficient hamster mutants UV5 and UVL-13

The cDNA sequence of the Chinese hamster xeroderma pigmentosum group D (CXPD) nucleotide excision repair gene was analyzed from three Chinese hamster ovary (CHO) cell lines: repair proficient strain AA8 and repair deficient, UV complementation group 2 strains UV5 and UVL-13. CXPD encodes a presumed ATP-dependent DNA helicase and is single copy in CHO lines due to the hemizygosity of chromosome 9. Comparison of the deduced wild-type AA8 CXPD protein sequence with that of the Chinese hamster V79 lung-derived cell line revealed two amino acid polymorphisms. Position 285 is glutamine in AA8 and arginine in V79, and position 298 is alanine in AA8 and threonine in V79. Comparison with the human XPD, Saccharomyces cerevisiae RAD3, and Schizosaccharomyces pombe rad15 homologs shows variability at these positions. Analysis of the CXPD sequence in the repair deficient CHO lines UV5 and UVL-13 revealed, in each case, a single base substitution resulting in an amino acid substitution. Position 116 is tyrosine in UV5 and cysteine in AA8, and the corresponding positions of XPD, RAD3, and rad15 are cysteine. Position 615 is glutamic acid in UVL-13 and glycine in AA8, and the corresponding positions of XPD, RAD3, and rad15 are glycine. In both UV5 and UVL-13, positions 285 and 298 are glutamine and alanine, respectively, as seen in AA8. These results suggest that cysteine 116 and glycine 615 are critical to the repair function of CXPD.

Molecular analysis of ERCC2 mutations in the repair deficient hamster mutants UVL-1 and V-H1

The cDNA sequence of the Chinese hamster ERCC2 nucleotide excision repair and transcription gene from the UVL-1 Chinese hamster ovary (CHO) mutant cell line and the V-H1 Chinese hamster V79 mutant line was analyzed. ERCC2 encodes a presumed ATP-dependent DNA helicase and is single copy in CHO lines due to the structural hemizygosity of chromosome 9. Both UVL-1 and V-H1 have intermediate levels of (6-4) photoproduct repair but are as highly UV sensitive as the group 2 mutants that have no detectable repair. Deficiency in cyclobutane dimer removal has also been shown for V-H1. In UVL-1, a single base substitution resulting in an Arg75-->Trp substitution in helicase domain Ia was identified. The equivalent amino acid position is also Arg in the human, mouse, Xiphophorus maculatus, Saccharomyces cerevisiae, and Schizosaccharomyces pombe homologs. In V-H1, a single base substitution resulting in a Thr46-->Ile substitution in helicase domain I (the ATP-binding domain) was identified in both alleles. The equivalent amino acid position is also Thr in the five homologs. Analysis of three V-H1 partial revertants revealed that they still have the original V-H1 mutation in both alleles, indicating that these are second site reversion events. Site-specific mutagenesis was used to introduce the Thr46-->Ile, Arg75-->Trp, and Lys48-->Arg (helicase domain I) mutations into a hamster ERCC2 expression plasmid. These plasmids each failed to confer UV resistance to group 2 mutant cells, further demonstrating that the changes identified are the causative mutations in V-H1 and UVL-1. Correlations between specific mutations, biochemical activities, and repair phenotype are discussed.