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Results for “DNA Excision Repair Protein ERCC-5”

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At least 19 recordsLinked to original sources
PubMed1999

Loss of heterozygosity and lack of mutations of the XPG/ERCC5 DNA repair gene at 13q33 in prostate cancer.

BACKGROUND: Three regions of chromosome 13 were previously identified for having loss of heterozygosity (LOH) in human prostate cancer. One of them, at 13q33, was defined by LOH at markers D13S158 and D13S280. The XPG/ERCC5 gene, a DNA repair gene that when mutated in the germline leads to xeroderma pigmentosum, has been mapped to 13q33, within one megabase of D13S158 and D13S280. This paper describes LOH and mutational analysis of the XPG gene in human prostate cancers, in order to determine whether the XPG gene is involved in the development of prostate cancer. METHODS: LOH of the XPG gene was analyzed in 40 primary prostate cancers and 14 metastases by using the microsatellite assay, and its mutations were examined in 5 cell lines, 14 metastases, and 8 tumors with LOH at 13q33 by using the single-strand conformation polymorphism (SSCP)-direct DNA sequencing analysis. RESULTS: Four of the 29 (14%) informative primary tumors and 4 of 8 (50%) metastases showed LOH for the XPG gene. Analysis of the 8 tumors with LOH at the 13q33 region, 14 metastases, and 5 cell lines of prostate cancer revealed two polymorphisms but no mutation of the gene. The polymorphism in exon 2 did not change the amino-acid sequence of the XPG protein, but the exon 15 polymorphism altered codon 1104 from histidine to aspartic acid. The two polymorphisms also occurred in individuals without prostate cancer. CONCLUSIONS: LOH at XPG in prostate cancer supports the conclusion that the 13q33 region contains a gene important in the development of prostate cancer, while lack of mutations of the gene suggests that XPG is not the target gene involved.

Adult↗
PubMed1998

PCNA binding through a conserved motif.

Proliferating cell nuclear antigen (PCNA) has recently been identified as a target for the binding of several proteins. The cell cycle regulatory protein, p21, and the replication endonuclease, Fen1, have already been described as competing for PCNA binding. Two recent reports have identified DNA (cytosine-5)methyltransferase (MCMT) and the DNA repair endonuclease XPG as binding to PCNA. The remarkable thing about these interactions is that they all seem to occur through a conserved motif that is likely to contact the same site on PCNA. This has fascinating implications for a regulatory network linking these diverse protein functions.

Amino Acid Sequence↗
PubMed2002

Expression of nucleotide excision repair genes and the risk for squamous cell carcinoma of the head and neck.

BACKGROUND: Phenotypic differences in the ability to repair genetic damage induced by tobacco carcinogens may reflect genetic differences in susceptibility to squamous cell carcinoma of the head and neck (SCCHN). The objective of this study was to assess the variation in baseline expression of five nucleotide excision repair genes between individuals with SCCHN and cancer free controls. METHODS: The authors conducted a hospital-based case-control study of 57 SCCHN patients and 105 cancer free controls. Using peripheral blood lymphocytes, a multiplex reverse transcriptase-polymerase chain reaction assay was used to quantitate in vitro the mRNA levels of five genes (ERCC1, XPB/ERCC3, XPG/ERCC5, CSB/ERCC6, and XPC) involved in the nucleotide excision repair pathway. RESULTS: The levels of ERCC1, XPB/ERCC3, XPG/ERCC5, and CSB/ERCC6 transcripts were lower in cases than in controls (P =0.0001, 0.096, 0.001, and 0.0001, respectively). In multivariate logistic regression analysis (adjusting for age, gender, race, smoking status, and alcohol use), low expression of ERCC1, XPB/ERCC3, XPG/ERCC5, and CSB/ERCC6 was associated with a statistically significant increased risk for SCCHN (adjusted odds ratios [95% confidence intervals] 6.42 [2.63-15.69], 2.86 [1.39-5.90], 3.69 [1.73-7.90], and 2.46 [1.19-5.09], respectively). CONCLUSIONS: Reduced expression of ERCC1, XPB/ERCC3, XPG/ERCC5, and CSB/ERCC6 is associated with a more than two-fold increased risk of SCCHN.

Adult↗
PubMed2001

Purkinje cell degeneration in mice lacking the xeroderma pigmentosum group G gene.

Laboratory mice carrying the nonfunctional xeroderma pigmentosum group G gene (the mouse counterpart of the human XPG gene) alleles have been generated by using gene-targeting and embryonic stem cell technology. Homozygote animals of this autosomal recessive disease exhibited signs and symptoms, such as postnatal growth retardation, reduced levels of activity, progressive ataxia and premature death, similar to the clinical manifestations of Cockayne syndrome (CS). Histological analysis of the cerebellum revealed multiple pyknotic cells in the Purkinje cell layer of the xpg homozygotes, which had atrophic cell bodies and shrunken nuclei. Further examination by an immunohistochemistry for calbindin-D 28k (CaBP) showed that a large number of immunoreactive Purkinje cells were atrophic and their dendritic trees were smaller and shorter than in wild-type littermates. These results indicated a marked degeneration of Purkinje cells in the xpg mutant cerebellum. Study by in situ detection of DNA fragmentation in the cerebellar cortex demonstrated that some deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin in situ nick labeling (TUNEL)-positive cells appeared in the granule layer of the mutant mice, but few cell deaths were confirmed in the Purkinje layer. These results suggested Purkinje cell degeneration in the mutant cerebellum was underway, in which much Purkinje cell death had not appeared, and the appearance of some abnormal cerebellar symptoms in the xpg-deficient mice was not only due to a marked Purkinje cell degeneration, but also to damage of other cells.

Animals↗
PubMed1996

Roles of XPG and XPF/ERCC1 endonucleases in UV-induced immunostaining of PCNA in fibroblasts.

To investigate the relationship between proliferating cell nuclear antigen (PCNA) complex formation and dual incisions in the nucleotide excision repair (NER) process, xeroderma pigmentosum group G (XP-G), XP-F, and XP-G equivalent mouse UV-sensitive mutant ERCC group 5 cells were utilized as a model in this study. These cells are deficient in endonucleases related to 3' (XP-G and ERCC group 5) or 5' (XP-F) incision of the DNA lesions in the NER process. PCNA complex formation was detected by an indirect immunofluorescence method after the cells were fixed in methanol. When Sps1 (XP-G) and XL216-7 (ERCC group 5) cells were UV irradiated, neither of them showed PCNA staining. In contrast, SFN4 (a human normal strain) and heterokaryons of Sps1 and XP96TO (XP-A) cells fused by polyethylene glycol treatment showed PCNA staining following UV irradiation. Furthermore, XLgfPAneo1 cells, derived from XL216-7 cells transfected with a plasmid containing mouse ERCC5 (xpg) cDNA, also restored staining and UV sensitivity. On the other hand, we observed a very faint PCNA staining in XP2YO (XP-F) cells, expressing no detectable ERCC1 or XPF protein, after UV irradiation. X rays induced PCNA staining in all cell lines with a similar staining pattern, and radiosensitivity was exactly the same between XL216-7 and XLgfPAneo1 cells. These results may have implications for the NER process in vivo in that coordinately occurring dual incisions by XPG and XPF/ERCC1 proteins play an important role in inducing PCNA complex formation, but the step may not be required for PCNA-dependent repair of X-ray-induced DNA damage.

Animals↗
PubMed1998

Complementation of transformed fibroblasts from patients with combined xeroderma pigmentosum-Cockayne syndrome.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are human hereditary disorders characterized at the cellular level by an inability to repair certain types of DNA damage. Usually, XP and CS are clinically and genetically distinct. However, in rare cases, CS patients have been shown to have mutations in genes that were previously linked to the development of XP. The linkage between XP and CS has been difficult to study because few permanent cell lines have been established from XP/CS patients. To generate permanent cell lines, primary fibroblast cultures from two patients, displaying characteristics associated with CS and belonging to XP complementation group G, were transformed with anorigin-of-replication-deficient simian virus 40 (SV40). The new cell lines, summation operatorXPCS1LVo- and summation operatorXPCS1ROo-,were characterized phenotypically and genotypically to verify that properties of the primary cells are preserved after transformation. The cell lines exhibited rapid growth in culture and were shown, by immunostaining, to express the SV40 T antigen. The summation operatorXPCS1LVo- and summation operatorXPCS1ROo- cell lines were hypersensitive to UV light and had an impaired ability to reactivate a UV-irradiated reporter gene. Using polymerase chain reaction (PCR) amplification and restriction enzyme cleavage, the summation operatorXPCS1ROo- cells were shown to retain the homozygous T deletion at XPG position 2972. This mutation also characterizes the parental primary cells and was evident in the XPG RNA. Finally, to characterize the XPG DNA repair deficiency in these cell lines, an episomal expression vector containing wild-type XPG cDNA was used to correct UV-induced damage in a beta-galactosidase reporter gene.

Cell Line, Transformed↗
PubMed1995

Complementary DNA sequence and chromosomal localization of xpg, the mouse counterpart of human repair gene XPG/ERCC5.

We have molecularly cloned and sequenced the mouse counterpart of the human repair gene XPG/ERCC5 cDNA. The mouse xpg cDNA had a single long open reading frame predicted to encode 1170 amino acid residues (predicted M(r) of 130,753). Northern blot analysis has been carried out to determine the size and tissue transcription specificity of the mouse xpg mRNA. The xpg gene expressed one species of transcript with 4.3 kb at similar levels in five mouse tissues examined. We have determined the chromosomal location of the xpg gene by both in situ hybridization and molecular linkage analysis. The xpg gene was localized at 2.3 cM proximal to the microsatellite locus D1Mit18 on the R-positive B band of mouse chromosome 1. By in situ hybridization with the mouse xpg probe, the rat homolog of the mouse xpg was localized on q22.3 band of rat chromosome 9, which has been known to have a conserved linkage homology to mouse chromosome 1. In the case of human, the XPG/ERCC5 gene has been reported to be assigned to human chromosome 13q32.3-q33.1, where any conserved linkage homology to mouse chromosome 1 has not been found so far. Thus, these results show new regions of conserved linkage homology among mouse chromosome 1, rat chromosome 9, and human chromosome 13q.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and structural analysis of the functional mouse genomic XPG gene.

The mouse XPG gene is a homolog of the human DNA excision repair gene known to be defective in the hereditary sun-sensitive disorder xeroderma pigmentosum (group-G). Defects in mouse XPG have been shown to directly affect the sensitivity of cultured cells to chemotherapy agents and may play a role in tumor cell drug resistance in vivo. A full-length cosmid clone of mouse XPG was isolated by complementation of the UV sensitivity and repair defect in CHO-UV135 cells. Exon mapping determined that the gene consisted of 15 exons within 32 kb of genomic DNA. Sequencing of intron-exon boundaries revealed that mouse XPG possesses a rare class of intron previously identified in only four other eukaryotic genes; it utilizes AT and AC dinucleotides instead of the expected GT and AG within the splice junctions. Promoter analysis determined that mouse XPG is expressed constitutively and probably initiates transcription from multiple start sites, yet, unlike the yeast homolog RAD2, we found no evidence that it is UVC inducible in cultured cells. Amino acid comparison with human XPG identified a highly conserved acidic region of homology not previously described.

Amino Acid Sequence↗
PubMed1995

XPG protein has a structure-specific endonuclease activity.

Biochemically active human DNA repair protein, xeroderma pigmentosum G (XPG), was overexpressed in insect cells by a recombinant baculovirus. The recombinant baculovirus produced XPG with a mobility of approximately 185 kDa in a denaturing polyacrylamide gel. Indirect immunofluorescence studies demonstrated that the recombinant full-length XPG protein was expressed predominantly as a nuclear protein. The recombinant XPG protein was purified to apparent homogeneity using Q-sepharose, S-300 size exclusion, and Mono Q column chromatography. XPG protein showed a structure-specific DNA endonuclease activity, and a preferential affinity to single-stranded DNA and RNA compared to double-stranded DNA.

Animals↗
PubMed1994

An ERCC5 gene with homology to yeast RAD2 is involved in group G xeroderma pigmentosum.

We have isolated a human excision repair gene ERCC5 which complements the defect of the mouse UV-sensitive mutant XL216 (rodent complementation group 5). Here we report cDNA cloning of human and mouse ERCC5 genes using an exon containing an ERCC5 fragment as a probe. The ERCC5 cDNA encodes a predicted 133-kDa nuclear protein that shares some homology with the product of the yeast DNA repair gene RAD2. Transfection with mouse ERCC5 cDNA restored normal levels of UV resistance to XL216 cells. Microinjection of ERCC5 cDNA specifically restored the defect of xeroderma pigmentosum group G cells (XP-G) as measured by unscheduled DNA synthesis, and XP-G cells stably transformed with ERCC5 cDNA showed nearly normal UV resistance.

Amino Acid Sequence↗
PubMed1996

Multiple nuclear localization signals in XPG nuclease.

We report here evidence for the mechanism of nuclear localization of XPG nuclease in human cells. Several candidate nuclear localization signal (NLS) peptides have been proposed for XPG protein. We have identified XPG peptides containing functional NLS and a potential nuclear retention signal (NRS) using in situ immunofluorescene localization of transiently expressed beta-galactosidase fusion proteins. Two XPG regions with putative NLS [amino acid (AA) coordinates: NLS-B (AA 1057-1074) and NLS-C (AA 1171-1185)] were each shown to independently localize the beta-gal extensively (> 80%) to the nucleus of HeLa cells. The C-terminus peptide containing NLS-C, an NLS conserved evolutionarily between yeasts and humans, also directed sub-localization of beta-galactosidase to intranuclear foci reminiscent of native XPG protein, as well as to peri-nucleolar regions. Peptides in the putative XPG 'NLS domain' (AA approximately 1051-1185) apparently function in concert for nuclear localization and also for retention of XPG in nuclear matrix-associated foci. Evidence presented elsewhere (Park et al., 1995) indicates that the peptide containing NLS-C (AA 1146-1185) also regulates the dynamic localization of XPG in the nucleus following UV-irradiation.

Amino Acid Sequence↗
PubMed2001

Phenotypes of Drosophila homologs of human XPF and XPG to chemically-induced DNA modifications.

DmXPF (mei9) and DmXPG (mus201) mutants are Drosophila homologs of the mammalian XPF and XPG genes, respectively. For Drosophila germ cells, causal correlations exist between the magnitude of a potentiating effect of a deficiency in these functions, measured as the M(NER-)/M(NER+) mutability ratio, and the type of DNA modification. M(NER-)/M(NER+) mutability ratios may vary with time interval between DNA adduct formation and repair, mutagen dose and depend also on the genetic endpoint measured. For forward mutations, there is no indication of any differential response of DmXPF compared to DmXPG. Subtle features appeared from a class-by-class comparison: (i) Methylating agents always produce higher M(NER-)/M(NER+) ratios than their ethylating analogs; (ii) M(NER-)/M(NER+) mutability ratios are significantly enhanced for cross-linking N-mustards, aziridine and di-epoxide compounds, but not for cross-linking nitrosoureas. The low hypermutability effects with bifunctional nitrogen mustards, aziridine and epoxide compounds are attributed to unrepaired mono-alkyl adducts; (iii) The efficient repair of mono-alkyl-adducts at ring nitrogens in wild-type germ cells is evident from the absence of a dose-response relationship for ethylene oxide, propylene imine and methyl methanesulfonate (MMS). These chemicals become powerful germline mutagens when the NER system is disrupted. Systematic studies of the type performed on germ cells are not available for somatic cells of Drosophila. The sparse data available show large differences in the response of germ cells and somatic cells. The bifunctional agent mechlorethamine (MEC) but not the monofunctional MMS or 2-chloroethylamine cause in NER(-) XXfemale symbol the highest potentiating effect on mitotic recombination. The causes of the discrepancy between the extraordinarily high activity of MEC in mus201 somatic cells and its low potentiating effect in germ cells is unknown at present.

Animals↗
PubMed2001

Factors influencing the removal of thymine glycol from DNA in gamma-irradiated human cells.

The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is > 80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before repair commences. However, if cells are irradiated with 0.25 Gy 4 h prior to the 2-Gy dose, removal of the thymine glycols commences immediately after the second irradiation, suggesting that repair of thymine glycol is inducible. Our current studies are directed at two aspects of the repair process, (1) factors involved in the repair process leading up to and including glycosylase-mediated removal of thymine glycol and (2) the control of the inducible response. We have observed that mutation of the XPG gene drastically reduced the level and rate of global removal of thymine glycol (induced by 2-Gy irradiation), and there was no evidence for an inducible response. Similar results were seen with a Cockayne syndrome B (CSB) cell line. We have also examined repair in quiescent and phytohemagglutinin-stimulated human lymphocytes. Both show similar kinetics for the rate of removal of thymine glycol under induced and noninduced conditions.

Animals↗
PubMed2000

Transcription-coupled repair of 8-oxoguanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome.

Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences. Defective TCR leads to a mutation frequency at 8-oxoG of 30%-40% compared to the normal 1%-4%. Surprisingly, unrepaired 8-oxoG blocks transcription by RNA polymerase II. These data imply that TCR is required for polymerase release to allow repair and that CS results from defects in TCR of oxidative lesions.

Cell Line↗
PubMed1999

The Drosophila ortholog of the human XPG gene.

Xeroderma pigmentosum complementation group G (XPG) protein is a junction-specific endonuclease which is indispensable for nucleotide excision repair (NER) of DNA in eukaryotes. Recent studies have hinted at a second, essential function for the XPG protein in higher eukaryotes. We undertook a comparison of the amino acid sequences of multiple XPG orthologs to determine if a motif or domain could be identified that is conserved uniquely in higher eukaryotes. A search of current databases allowed us to retrieve complete amino acid sequences for the human, mouse and Xenopus XPG proteins, and for two yeast orthologs. We also identified an incomplete Drosophila open reading frame (ORF) that was a good candidate for the XPG protein. We cloned a complete Drosophila cDNA for this ORF and examination of the primary amino acid sequence suggests that this cDNA encodes the Drosophila ortholog of XPG. A comparison of all six orthologous polypeptides reveals the presence of two previously unidentified conserved domains. One of these is unique to all four higher eukaryotic sequences. Conceivably this domain evolved to support the essential function of XPG protein.

Amino Acid Sequence↗