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Query: UNIPROT:P06889 (Mol)
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Complementation of DNA excision repair defect in xeroderma pigmentosum cells of group C (XP-C) has been achieved by the transfer of human chromosome 5. Individual human chromosomes tagged with a selectable marker were transferred to XP-C cells by microcell fusion from mouse-human hybrid cell lines each bearing a single different human chromosome. Analysis of the chromosome transfer clones revealed that introduction of chromosome 5 into XP-C cells corrected the DNA repair defect as well as UV-sensitive phenotypes, while chromosomes 2, 6, 7, 9, 13, 15, 17, and 21 failed to complement. The introduced chromosome 5 in complemented UVr clones was distinguished from the parental XP-C chromosomes by polymorphism for dinucleotide (CA)n repeats at two loci, D5S117 and D5S209. In addition, an intact marked chromosome 5 was rescued into mouse cells from a complemented UVr clone by microcell fusion. Five subclones of a complemented clone that had lost the marked chromosome 5 exhibited UV-sensitive and repair-deficient phenotypes identical to parental XP-C cells. Concordant loss of the transferred chromosome and reappearance of XP-C phenotype further confirmed the presence of a DNA repair gene on human chromosome 5.
Somat Cell Mol Genet 1993 Jan
PMID:Complementation of DNA repair defect in xeroderma pigmentosum cells of group C by the transfer of human chromosome 5. 846 Apr 1

In the accompanying paper we demonstrated that endonuclease III-sensitive sites in the MAT alpha and HML alpha loci of Saccharomyces cerevisiae are repaired by the Nucleotide Excision Repair (NER) pathway. In the current report we investigated the repair of endonuclease III sites, 6-4 photoproducts and cyclobutane pyrimidine dimers (CPDs) in a rad14-2 point mutant and in a rad14 deletion mutant. The RAD14 gene is the yeast homologue of the human gene that complements the defect in cells from xeroderma pigmentosum (XP) patients belonging to complementation group A. In the point mutant we observed normal repair of endonuclease III site (i.e. as wild type), but no removal of CPDs at the MAT alpha and HML alpha loci. Similar experiments were undertaken using the recently created rad14 deletion mutant. Here, neither endonuclease III sites nor CPDs were repaired in MATa or HMRa. Thus the point mutant appears to produce a gene product that permits the repair of endonuclease III sites, but prevents the repair of CPDs. Previously it was found that in the genome overall, repair of 6-4 photoproducts was less impaired that repair of CPDs in the point mutant. The deletion mutant repairs neither CPDs nor 6-4 photoproducts in the genome overall. This finding is consistent with the RAD14 protein being involved in lesion recognition in yeast. A logical interpretation is that the rad14-2 point mutant produces a modified protein that enables the cell to repair endonuclease III sites and 6-4 photoproducts much more efficiently than CPDs. This modified protein may aid studies designed to elucidate the role of the RAD14 protein in lesion recognition.
Mol Gen Genet 1996 Mar 07
PMID:The levels of repair of endonuclease III-sensitive sites, 6-4 photoproducts and cyclobutane pyrimidine dimers differ in a point mutant for RAD14, the Saccharomyces cerevisiae homologue of the human gene defective in XPA patients. 860 69

Gene-specific DNA damage levels were determined by quantitative polymerase chain reaction (QPCR) after treating cytochrome P450 (CYP) 1A1-expressing xeroderma pigmentosum fibroblasts with [3H]benzo[a]pyrene-trans-7,8-dihydrodiol ([3H]BPD) or [3H]benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide ([3H]BPDE). DNA damage in the p53 gene (which is transcriptionally active) and the beta-globin gene (which is transcriptionally inactive) was measured in cells treated with [3H](+/-)-anti-BPDE, [3H](+/-)-BPD, and [3H](-)-BPD. DNA adduct formation in the genome overall was determined by measuring the incorporation of 3H into DNA. DNA damage in a p53 gene fragment (exons 8-9, 445 bp) was readily detected by QPCR. DNA damage was either not detected or much reduced in a similarly sized target in the beta-globin gene (exons 1-2, 551 bp). At equivalent levels of genomic DNA adducts, BPD treatment induced more damage in the p53 gene than BPDE treatment did. The lesion frequencies in the p53 and beta-globin genes in purified DNA treated with BPDE in vitro were the same, indicating that there was no sequence-specific basis for preferential lesion formation in the p53 gene in treated cells. DNA damage in both the p53 and beta-globin genes showed a dose response to [3H](-)-BPD. The frequency of BPD-induced lesions in the p53 gene was sixfold to sevenfold greater than in the beta-globin gene and 200- to 300-fold greater than in bulk DNA. The BPD-induced lesion frequency in the beta-globin gene was 30- to 50-fold greater than in bulk DNA. The data indicate that the distribution of BPDE-induced DNA lesions is dramatically nonrandom and suggest that the nonrandomness is governed by DNA sequence composition, chromatin structure, and dose rate.
Mol Carcinog 1996 May
PMID:Preferential DNA damage in the p53 gene by benzo[a]pyrene metabolites in cytochrome P4501A1-expressing xeroderma pigmentosum group A cells. 863 92

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.
Mol Cell Biol 1996 Nov
PMID:ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs. 888 84

Roberts syndrome (RS) is a rare, recessively inherited disorder characterized by growth retardation, limb reductions and craniofacial deformities. Cells from a subset of afflicted individuals, termed RS+, display unusual separation or puffing of the heterochromatic regions of their chromosomes and are hypersensitive to several DNA-damaging agents including mitomycin C (MMC) and cisplatin, both of which can induce interstrand crosslinks in DNA. For this reason, we have investigated the ability of RS+ fibroblasts to repair cisplatin-induced DNA lesions using adenoviris as a probe. Host cell reactivation of cisplatin-treated adenovirus (Ad) was significantly reduced in nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) cells but was normal in the two RS+ fibroblast strains and the Fanconi's anemia (FA)fibroblast strain tested. The capacity of cisplatin-treated cells for Ad DNA synthesis was reduced in XP and FA cells compared to normal human cells, but was not reduced in RS+ cells. These results indicate that the hypersensitivity of RS+ cells to cisplatin is not due to a deficiency in NER nor due to a deficiency in the pathway which leads to cisplatin hypersensitivity in FA cells. It is possible that the abnormal heterochromatin organisation of RS+ cells selectively renders the heterochromatic regions of the genome more susceptible to mutagen damage and/or less available for repair.
Somat Cell Mol Genet 1996 Sep
PMID:Roberts syndrome fibroblasts showing cisplatin hypersensitivity have normal host cell reactivation of cisplatin-treated adenovirus and normal capacity of cisplatin-treated cells for adenovirus DNA synthesis. 903 48

Defects in the xeroderma pigmentosum complementation group A-correcting (XPA) gene, which encodes a component of the nucleotide excision repair (NER) pathway, are associated with the cancer-prone human disease xeroderma pigmentosum. We previously generated mice lacking the XPA gene, which develop normally but are highly sensitive to ultraviolet-B and 7,12-dimethylbenz[a] anthracene-induced skin tumors. Here we report that XPA-deficient mice spontaneously developed hepatocellular adenomas at a low frequency as they aged. Furthermore, oral treatment of XPA-deficient mice with the carcinogen benzo[a]pyrene (B[a]P) resulted in the induction of mainly lymphomas. These tumors appeared earlier and with a higher incidence than in B[a]P-treated wild-type and heterozygous mice. Our results show for the first time that XPA-deficient mice also displayed an increased sensitivity to developing tumors other than tumors of the skin.
Mol Carcinog 1997 May
PMID:Spontaneous liver tumors and benzo[a]pyrene-induced lymphomas in XPA-deficient mice. 918 Sep 28

We demonstrated previously that the nucleoside of fludarabine (F-ara-A), a clinically effective agent against chronic lymphocytic leukemia and low-grade lymphoma, produces synergistic cytotoxicity against cisplatin-resistant CP2.0 human colon tumor cells when administered in combination with cisplatin. The purpose of this study was 2-fold: (i) to determine whether the synergy occurs in K562 human chronic myelogenous leukemia cells, which, unlike CP2.0 cells, are relatively resistant to drug-induced apoptosis because they express P210(bcr-abl) and (ii) to study the underlying mechanism for the synergy if the enhancement of cytotoxicity occurs in K562 cells. When K562 cells were treated with fludarabine nucleoside and cisplatin as single agents for 4 hr, IC50 values for fludarabine and cisplatin were 3.33 and 2.28 microM, respectively, as measured by a clonogenic survival assay. The simultaneous treatment of K562 cells with the two agents resulted in synergistic cell killing as determined by median-effect analysis. Such synergistic cell killing by combined cisplatin and fludarabine could not be detected in repair-deficient human xeroderma pigmentosum cell lines. Within the range of cytotoxic concentrations, fludarabine (2.5-15 microM) and cisplatin (3-30 microM) as single agents produced no detectable internucleosomal DNA fragmentation as revealed by gel electrophoresis, nor did the combination of the two drugs induce apoptotic DNA degradation. The effects of fludarabine on the repair of cisplatin-induced DNA adducts and interstrand cross-links in K562 cells were analyzed to determine their correlation with the cytotoxic synergy. The interstrand cross-links were measured by the ethidium bromide binding fluorescence assay and quantitative Southern blotting technique. Repair of the intrastrand adducts was detected with whole-cell extracts using a cisplatin-damaged plasmid as the substrate for the in vitro repair assay. Fludarabine at clinically achievable concentrations (1.5-4.5 microM fludarabine nucleoside; 20-100 microM fludarabine triphosphate) inhibited the repair of the DNA lesions induced by cisplatin in a dose-dependent fashion in K562 cells but not in xeroderma pigmentosum cells. Cotreatment with fludarabine preferentially increased the number of interstrand cross-links induced by cisplatin in actively transcribed genes in K562 cells. These data demonstrate the DNA-repair-inhibitory effect of fludarabine and suggest that this effect may contribute to the synergistic cytotoxicity of the fludarabine/cisplatin combination that resulted in decreased clonogenic survival of apoptosis-resistant K562 cells.
Mol Pharmacol 1997 Nov
PMID:Fludarabine-mediated repair inhibition of cisplatin-induced DNA lesions in human chronic myelogenous leukemia-blast crisis K562 cells: induction of synergistic cytotoxicity independent of reversal of apoptosis resistance. 935 70

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.
Mol Cell Biol 1997 Dec
PMID:Identification and characterization of XPC-binding domain of hHR23B. 937 23

Nucleotide excision repair (NER) is a process required to remove DNA damage inflicted upon our skin by the short-wave bands of natural sunlight. Defective NER may result in a high risk of UV-induced skin tumors, since it occurs in patients with the inherited disorder xeroderma pigmentosum (XP). However, Cockayne's syndrome (CS) and PIBIDS (a photosensitive form of trichothiodystrophy) are also disorders with defective NER, but show no evidence of an elevated risk of cancer. In addition, many of CS and PIBIDS symptoms are difficult to explain on the basis of an NER defect only. Recent new insights into the molecular mechanisms of NER have shown additional involvements of many NER enzymes in other cellular processes. These multiple functions are likely to be the basis of the complex symptomatology of XP, CS and PIBIDS. Specific gene-targeted mouse models will probably help to solve these intricacies.
Cytokines Mol Ther 1996 Jun
PMID:Multiple involvement of nucleotide excision repair enzymes: clinical manifestations of molecular intricacies. 938 96

Trichothiodistrophy (TTD), xeroderma pigmentosum (XP), and Cockayne's syndrome (CS) are three distinct human diseases with sensitivity to ultraviolet (UV) radiation affected by mutations in genes involved in nucleotide excision repair (NER). Among the many responses of human cells to UV irradiation, both nuclear accumulation of p53, a tumor suppressor protein, and alterations in cell-cycle checkpoints play crucial roles. The purpose of this study was to define the signals transmitted after UV-C-induced DNA damage, which activates p53 accumulation in TTD/XP-D fibroblasts, and compare this with XP-D cell lines that carry different mutations in the same gene, XPD. Our results showed that p53 was rapidly induced in the nuclei of TTD/XP-D and XP-D fibroblasts in a dose-dependent manner after UV-C irradiation, as seen in XP-A and CS-A fibroblasts, much lower doses being required for the protein accumulation than in normal human fibroblasts, XP variant cells, and XP-C cells. The kinetics of accumulation of p53 and two effector proteins involved in cell-cycle arrest, WAF1 and GADD45, were also directly related to the repair potential of the cells, as in normal human fibroblasts their levels declined after 24 h, the time required for repair of UV-induced lesions, whereas NER-deficient TTD/XP-D cells showed p53, WAF1, and GADD45 accumulation for over 72 h after irradiation. Our results indicate that p53 accumulation followed by transcriptional activation of genes implicated in growth arrest is triggered in TTD/XP-D cells by the persistence of cyclobutane pyrimidine dimers, which are known to block transcription, on the transcribed strands of active genes.
Mol Carcinog 1997 Dec
PMID:Prolonged p53 protein accumulation in trichothiodystrophy fibroblasts dependent on unrepaired pyrimidine dimers on the transcribed strands of cellular genes. 943 78


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