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PubMed1975

Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation.

Cells cultured from most patients suffering from the sunlight-sensitive hereditary disorder xeroderma pigmentosum are defective in the ability to excise ultraviolet light (UV)-induced pyrimidine dimers from their DNA. There is, however, one class of these patients whose cells are completely normal in this excision repair process. We have found that these cells have an abnormality in the manner in which DNA is synthesized after UV-irradiation. The time taken to convert initially low-molecular-weight DNA synthesized in UV-irradiated cells into high-molecular-weight DNA similar in size to that in untreated cells is much greater in these variants than in normal cells. Furthermore, this slow conversion of low to high-molecular-weight newly synthesized DNA is drastically inhibited by caffeine, which has no effect in normal cells. Two cell lines from classes of xeroderma pigmentosum that are defective in excision-repair show intermediate effects, with regard to both the time taken to convert newly synthesized DNA to high molecular weight and the inhibition of this process by caffeine.

Caffeine↗

PubMed1997

Decoupling of DNA excision repair and RNA transcription in translocation breaksite regions of plasmacytoma-susceptible BALB/cAnPt mice.

Preferential repair of pyrimidine dimers in rodent cells is thought to be directly coupled to the RNA transcription machinery. The most compelling evidence for this notion is the finding that excision repair occurs more rapidly in the template strand of DNA of transcribed genes than in the non-template strand. A thorough test of this coupling concept by careful comparison of the rate of repair to the rate of transcription of a gene and its regulatory region has not been reported. In the present study, we used nuclear run-on as a measure of transcription in the c-myc and Pvt1 genes in normal B-lymphoblasts from plasmacytoma-susceptible (BALB/cAnPt) and plasmacytoma-resistant (DBA/2N) strains of mice. Previous studies have shown that these loci, but not c-abl or Dhfr are repaired differently in mouse strains: poorly in BALB/cAnPt but efficiently in DBA/2N. The results presented here indicate that in DBA/2N cells, run-on transcription from both DNA strands can be readily detected in the regions of c-myc and Pvt1 that were efficiently repaired. Unexpectedly, however, in BALB/cAnPt lymphoblasts, transcription was equivalent to that of DBA/2N, despite a dramatic reduction in efficiency of excision repair. This finding indicates that, in BALB/cAnPt lymphoblasts, DNA repair 5' to c-myc and in Pvt1 is decoupled from the RNA transcription machinery. We postulate that this dissociation of repair and transcription represents a BALB/cAnPt-specific defect in a component of the transcription/repair complex that specifically compromises repair activity but not transcription. This defect may be responsible for the inability of normal BALB/cAnPt lymphoblasts to repair DNA sequences in the c-myc 5' flank and the Pvt1 gene, inducing gene-specific instability that predisposes these loci to genetic accidents, including chromosomal translocation, retroviral integration and other mutations.

Animals↗

PubMed1998

The yeast RAD7 and RAD16 genes are required for postincision events during nucleotide excision repair. In vitro and in vivo studies with rad7 and rad16 mutants and purification of a Rad7/Rad16-containing protein complex.

In eukaryotes, nucleotide excision repair (NER) is a complex reaction requiring multiple proteins. In the yeast Saccharomyces cerevisiae, two of these proteins, Rad7 and Rad16, are specifically involved in the removal of lesions from transcriptionally silent regions of the genome in vivo. Extracts prepared from rad7 or rad16 mutant cells are deficient, but not totally defective, in both oligonucleotide excision and repair synthesis of damaged plasmid DNA. We show that these extracts are, however, fully proficient in the incision step of the NER reaction in vitro. Furthermore, using a cdc9 mutant to trap incision intermediates, we demonstrate that rad7 and rad16 mutants are proficient in NER-dependent DNA incision in vivo. A purified protein complex containing both Rad7 and Rad16 proteins complements the oligonucleotide excision and repair synthesis defects in rad7 and rad16 mutant extracts. We conclude that the products of the RAD7 and RAD16 genes are involved in a postincision event(s) during NER in yeast.

Adenosine Triphosphatases↗

PubMed1982

Effects of DNA replication inhibitors on UV excision repair in synchronised human cells.

When HeLa cells are irradiated with UV and treated with the DNA synthesis inhibitors hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), DNA strand breaks accumulate at sites where excision repair of DNA damage has been inhibited after the incision step. This break accumulation occurs in mitotic, G1 and S phase cells. But UV-induced repair synthesis of DNA, as measured by [3H]thymidine incorporation into unreplicated DNA, is not inhibited by HU and ara C in G1 or S phase cells, even though replicative synthesis is virtually abolished. Repair and replication must therefore utilise different DNA precursor pools, or different DNA synthetic systems; and the action of Hu and ara C in causing strand break accumulation may occur at the ligation step of excision repair.

Cytarabine↗

PubMed2005

Reduced excision repair cross-complementing 1 expression associates with enhanced papilloma formation and fibroblast transformation after genetic disruption of secreted protein acidic and rich in cysteine.

SPARC (secreted protein acidic and rich in cysteine)/ osteonectin/BM-40 is a matricellular protein implicated in development, cell transformation and tumorigenesis. We have examined the role of SPARC in cell transformation induced chemically with 7,12-dimethylbenz[a]anthracene (DMBA) and 12-tetradecanoylphorbol-13-acetate (TPA) in embryonic fibroblasts and in the skin of mice. Embryonic fibroblasts from SPARCnull mice showed increases in cell proliferation, enhanced sensitivity to DMBA and a higher number of DMBA/TPA-induced transformation foci. The number of DMBA-DNA adducts was 9 times higher in SPARCnull fibroblasts and their stability was lower than wild-type fibroblasts, consistent with a reduction in excision repair cross-complementing 1 the nucleotide excision repair enzyme in these cells. The SPARCnull mice showed an increase in both the speed and number of papillomas forming after topical administration of DMBA/TPA to the skin. These papillomas showed reduced growth and reduced progression to a more malignant phenotype, indicating that the effect of SPARC on tumorigenesis depends upon the transformation stage and/or tissue context. These data reinforce a growing number of observations in which SPARC has shown opposite effects on different tumor types/stages.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1973

Characterization of excision repair in Neurospora crassa.

The excision of pyrimidine dimers from the deoxyribonucleic acid (DNA) of Neurospora crassa was examined. Postirradiation incubation in the presence of several chemicals known to inhibit various repair systems indicated that caffeine reduced the rate of excision twofold, but did not inhibit excision completely as did proflavine and quinacrine. Examination of the time course of excision showed that repair occurs at a relatively rapid rate: approximately 60 dimers excised per min after 500 ergs/mm(2). Further evidence for rapid excision was obtained by sedimentation analysis of DNA; the maximal number of breaks introduced during repair was three, suggesting that breaks are repaired almost as fast as they are made and that only a few dimers are repaired at a time. Repair synthesis was measured by prelabeling the DNA with (15)N and D(2)O, and then subjecting the DNA to equilibrium density gradient centrifugation after postirradiation incubation with (32)P. Accumulation of single-strand breaks with increasing dose of ultraviolet radiation suggested that the limiting step was subsequent to the incision and excision steps of repair. Equilibrium CsCl centrifugation demonstrated that the limiting step in excision was repair synthesis.

Acridines↗

PubMedDate not supplied

Inhibition of excision repair without influence upon UV-sensitivity and UV-mutability in Escherichia coli B/r Hcr+.

Pre-irradiation starvation of exponentially growing Escherichia coli B/r Hcr+ thy-try- strain for thymine and tryptophan causes inhibition of pyrimidine dimer excision from ultraviolet damaged cells DNA. This inhibition of excision repair has not resulted in increasing ultraviolet sensitivity nor in increasing frequency of ultraviolet induced tryptophan revertants. The possible mechanisms of the non-excision repair in the prestarved cells, which is at least as accurate and effective as the whole dark repair in exponentially growing cells, are discussed.

DNA Repair↗

PubMed1997

Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts.

We have shown previously that Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in the removal of UV-induced cyclobutane pyrimidine dimers from genomic DNA, but still proficient in the transcription-coupled repair pathway (Ford, J. M., and Hanawalt, P. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8876-8880). We have now utilized monoclonal antibodies specific for cyclobutane pyrimidine dimers or 6-4 photoproducts, respectively, to measure their repair in UV-irradiated human fibroblasts. Cells homozygous for p53 mutations were deficient in the repair of both photoproducts, whereas cells heterozygous for mutant p53 exhibited normal repair of 6-4 photoproducts, but decreased initial rates of removal of cyclobutane pyrimidine dimers, compared with normal cells. The specificity of the effect of wild-type p53 on nucleotide excision repair was demonstrated in a p53 homozygous mutant cell line containing a tetracycline-regulated wild-type p53 gene. Wild-type p53 expression and activity were suppressed in the presence of tetracycline, whereas withdrawal of tetracycline resulted in the induction of p53 expression, cell cycle checkpoint activation, and DNA damage-induced apoptosis. The regulated expression of wild-type p53 resulted in the recovery of normal levels of repair of both cyclobutane pyrimidine dimers and 6-4 photoproducts in genomic DNA, but did not alter the transcription-coupled repair of cyclobutane pyrimidine dimers. Therefore, the wild-type p53 gene product is an important determinant of nucleotide excision repair activity in human cells.

Cells, Cultured↗

PubMed1988

The role of the excision-repair enzymes in mutation-induction by cis-Pt(NH3)2Cl2.

Mutation induction by cis-Pt(NH3)2Cl2 (cisplatin) has been shown to be absent in E.coli strains carrying a deletion of the uvrB gene (1). This suggested that excision-repair, which is normally thought to be error-free, is involved in mutation induction with cisplatin. Here, the role of the excision repair enzymes UvrA, UvrB and UvrC is investigated using E.coli strains with different repair capacities. It is shown that cisplatin induced mutagenesis is dependent both on UvrA and UvrB but not on UvrC. Of the UvrB enzyme the N-terminal 113 aminoacids are sufficient for mutation induction by cisplatin.

Cisplatin↗

PubMed1986

Pyrimidine dimer excision repair of DNA in Bacteroides fragilis wild-type and mitomycin C-sensitive/UV-sensitive mutants.

An enzyme preparation purified from Micrococcus luteus was shown to be specific for UV-induced pyrimidine dimers and was suitable for the detection of DNA excision repair systems. The wild-type Bacteroides fragilis Bf-2 strain and a mitomycin C-sensitive mutant (MTC25) had constitutive dimer excision systems which functioned efficiently under anaerobic and aerobic conditions. A UV-sensitive mutant (UVS9) had markedly reduced levels of the constitutive dimer excision systems under anaerobic and aerobic conditions. Since liquid holding recovery under aerobic conditions was inhibited by chloramphenicol whereas the final level of excision repair in B. fragilis Bf-2 was not affected, it is concluded that pyrimidine dimer removal is not the process responsible for increased physiological aerobic liquid holding recovery.

Bacteroides fragilis↗

PubMed1990

Transcription preferentially inhibits nucleotide excision repair of the template DNA strand in vitro.

It has been reported that pyrimidine dimers (pyrimidine mean value of pyrimidine) are removed preferentially from actively transcribing genes. Furthermore, the preferential repair is restricted to the transcribed strand of these genes. Currently there is no mechanistic explanation for these phenomena. In this study we investigated the effect of transcription on nucleotide excision repair using defined Escherichia coli systems consisting of DNA substrates containing a strong promoter and either (a) a T mean value of T at a defined position in the nontranscribed or transcribed strand or (b) photoproducts randomly distributed in both strands, as well as transcription and nucleotide excision repair enzymes. While a T mean value of T in the nontranscribed strand had no effect on transcription, a photodimer in the transcribed strand blocked transcription causing RNA polymerase to stall at the T mean value of T site. This stalled elongation complex inhibited the excision of the photodimer by (A)BC excinuclease resulting in a net effect of preferential repair of the nontranscribed strand in a mixture containing both substrates. Similarly, when we conducted transcription/repair experiments with a superhelical plasmid no enhanced repair of the transcribed gene was observed compared to nontranscribed regions. We conclude that RNA polymerase stalled at a photodimer does not direct the (A)BC excinuclease to the damaged template strand and therefore cannot account for the strand-specific repair observed in vivo.

Base Sequence↗

PubMed1992

Enhanced excision repair activity in mammalian cells after ionizing radiation.

Monkey CV-1 cells which had received 5 Gy 12 h before harvesting lysates from their cell cultures contained approximately three times as much DNA excision repair enzyme activity as unirradiated cells. The activity was determined in crude cell lysates by the release of intermediate mobility DNA fragments and fragments with 3'-phosphoryl ends from 5'-32P-end labelled irradiated 95 bp alpha DNA. Different 3'-termini endow the fragments with differing mobilities, signifying steps in the processing of radiation damaged DNA. Similar results were obtained when Krebs II mouse tumour cells growing in mice as ascites received 5 Gy 12 h before harvest. The enzyme activities from CV-1 cells and from Krebs II cells were partially purified as 60-70 kDa proteins on Superose 12 or Ultrogel AcA-54 columns. Divalent cations were not required for enzyme activity. A 23 nucleotide long defined duplex oligodeoxynucleotide substrate containing a single 8-oxodG residue was also very actively cleaved by the partially purified cell enzymes. 8-oxoguanine is a major product of ionizing radiation's action on DNA and was recognized by the enzymes described here. The mechanism by which radiation increased excision repair activity of cellular enzymes is not understood.

Animals↗

PubMed1994

Mutation spectra of 1,2-dibromoethane, 1,2-dichloroethane and 1-bromo-2-chloroethane in excision repair proficient and repair deficient strains of Drosophila melanogaster.

DNA sequence changes produced by 1,2-dibromoethane (DBE), 1,2-dichloroethane (DCE) and 1-bromo-2-chloroethane (BCE) were analyzed using the vermilion locus of Drosophila melanogaster. Under excision repair proficient (exr+) conditions (mutagenized exr+ males mated with exr+ females) all mutants isolated from the first generation (F1) after DBE and DCE exposure represented DNA rearrangements (multi-locus deletions, small deletions with tandem repeats, duplicate insertions). By contrast, mutants expressing a vermilion phenotype only in the F2 (F1 mosaics) all carried single bp changes. When exr+ males, after exposure to DBE, were mated to excision repair deficient (exr-) mus 201 females 11 of 14 mutational events isolated from either F1 or F2 progeny were single bp changes. In general the mutation spectra for the three dihaloalkanes were similar to the spectrum obtained at the same locus for the direct-acting monofunctional agent methylmethanesulfonate (MMS). The data lend support to the conclusions that these 1,2-dihaloalkanes are genotoxic through modification at ring nitrogens in DNA, primarily at the N7 of guanine and, to a lesser extent, at the N1 of adenine. These N-adducts could be directly miscoding. However, more important for the mutagenic action of the chemicals seems to be the formation of non-coding lesions and/or misrepair.

Animals↗

PubMed1994

DNA polymerase delta is required for base excision repair of DNA methylation damage in Saccharomyces cerevisiae.

We present evidence that DNA polymerase delta of Saccharomyces cerevisiae, an enzyme that is essential for viability and chromosomal replication, is also required for base excision repair of exogenous DNA methylation damage. The large catalytic subunit of DNA polymerase delta is encoded by the CDC2(POL3) gene. We find that the mutant allele cdc2-2 confers sensitivity to killing by methyl methanesulfonate (MMS) but allows wild-type levels of UV survival. MMS survival of haploid cdc2-2 strains is lower than wild type at the permissive growth temperature of 20 degrees C. Survival is further decreased relative to wild type by treatment with MMS at 36 degrees C, a nonpermissive temperature for growth of mutant cells. A second DNA polymerase delta allele, cdc2-1, also confers a temperature-sensitive defect in MMS survival while allowing nearly wild-type levels of UV survival. These observations provide an in vivo genetic demonstration that a specific eukaryotic DNA polymerase is required for survival of exogenous methylation damage. MMS sensitivity of a cdc2-2 mutant at 20 degrees C is complemented by expression of mammalian DNA polymerase beta, an enzyme that fills single-strand gaps in duplex DNA in vitro and whose only known catalytic activity is polymerization of deoxyribonucleotides. We conclude, therefore, that the MMS survival deficit in cdc2-2 cells is caused by failure of mutant DNA polymerase delta to fill single-strand gaps arising in base excision repair of methylation damage. We discuss our results in light of current concepts of the physiologic roles of DNA polymerases delta and epsilon in DNA replication and repair.

Alleles↗

PubMed2004

Inactivating mutations of the human base excision repair gene NEIL1 in gastric cancer.

Oxidized DNA base lesions, such as thymine glycol (Tg) and 8-hydroxyguanine, are often toxic and mutagenic and have been implicated in carcinogenesis. To clarify whether NEIL1 protein, which exhibits excision repair activity towards such base lesions, is involved in gastric carcinogenesis, we examined 71 primary gastric cancers from Japanese patients and four gastric cancer cell lines for mutations and genetic polymorphisms of the NEIL1 gene. We also examined 20 blood samples from Chinese patients for NEIL1 genetic polymorphisms. Three mutations (c.82_84delGAG:p.Glu28del, c.936G > A and c.1000A > G:p.Arg334Gly) and two genetic polymorphisms were identified. When the excision repair activity towards double-stranded oligonucleotide containing a Tg:A base pair was compared among six types of recombinant NEIL1 proteins, p.Glu28del-type NEIL1, found in a primary case, was found to exhibit an extremely low activity level. Moreover, c.936G > A, located in the last nucleotide of exon 10 and detected in the KATO-III cell line, was shown to be associated with a splicing abnormality using an in vivo splicing assay. An immunofluorescence analysis showed that the wild-type NEIL1 protein, but not the truncated protein encoded by the abnormal transcript arising from the c.936G > A mutation, was localized in the nucleus, suggesting that the truncated protein is unlikely to be capable of repairing nuclear DNA. An expression analysis revealed that NEIL1 mRNA expression was reduced in six of 13 (46%) primary gastric cancer specimens that were examined. These results suggest that low NEIL1 activities arising from mutations and reduced expression may be involved in the pathogenesis in a subset of gastric cancers.

Alternative Splicing↗

PubMed2004

XRCC1-DNA polymerase beta interaction is required for efficient base excision repair.

X-ray repair cross-complementing protein-1 (XRCC1)-deficient cells are sensitive to DNA damaging agents and have delayed processing of DNA base lesions. In support of its role in base excision repair, it was found that XRCC1 forms a tight complex with DNA ligase IIIalpha and also interacts with DNA polymerase beta (Pol beta) and other base excision repair (BER) proteins. We have isolated wild-type XRCC1-DNA ligase IIIalpha heterodimer and mutated XRCC1-DNA ligase IIIalpha complex that does not interact with Pol beta and tested their activities in BER reconstituted with human purified proteins. We find that a point mutation in the XRCC1 protein which disrupts functional interaction with Pol beta, affected the ligation efficiency of the mutant XRCC1-DNA ligase IIIalpha heterodimer in reconstituted BER reactions. We also compared sensitivity to hydrogen peroxide between wild-type CHO-9 cells, XRCC1-deficient EM-C11 cells and EM-C11 cells transfected with empty plasmid vector or with plasmid vector carrying wild-type or mutant XRCC1 gene and find that the plasmid encoding XRCC1 protein, that does not interact with Pol beta has reduced ability to rescue the hydrogen peroxide sensitivity of XRCC1- deficient cells. These data suggest an important role for the XRCC1-Pol beta interaction for coordinating the efficiency of the BER process.

Animals↗

PubMed2002

p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts.

Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the maximum adduct levels achieved. The fact that this p53-dependent effect is noted at levels of DNA adducts that are commonly found in human tissues (i.e., <100 adducts/10(8) nucleotides) because of environmental factors such as smoking is particularly significant with respect to human carcinogenesis related to environmental exposure.

Carcinogens, Environmental↗

PubMed2005

Investigations of pyrimidine dimer glycosylases--a paradigm for DNA base excision repair enzymology.

The most prevalent forms of cancer in humans are the non-melanoma skin cancers, with over a million new cases diagnosed in the United States annually. The portions of the body where these cancers arise are almost exclusively on the most heavily sun-exposed tissues. It is now well established that exposure to ultraviolet light (UV) causes not only damage to DNA that subsequently generates mutations and a transformed phenotype, but also UV-induced immunosuppression. Human cells have only one mechanism to remove the UV-induced dipyrimidine DNA photoproducts: nucleotide excision repair (NER). However, simpler organisms such as bacteria, bacteriophages and some eukaryotic viruses contain up to three distinct mechanisms to initiate the repair of UV-induced dipyrimidine adducts: NER, base excision repair (BER) and photoreversal. This review will focus on the biology and the mechanisms of DNA glycosylase/AP lyases that initiate BER of cis-syn cyclobutane pyrimidine dimers. One of these enzymes, the T4 pyrimidine dimer glycosylase (T4-pdg), formerly known as T4 endonuclease V has served as a model in the study of this entire class of enzymes. It was the first DNA repair enzyme: (1) for which a biologically significant processive nicking activity was demonstrated; (2) to have its active site determined, (3) to have its crystal structure solved, (4) to be shown to carry out nucleotide flipping, and (5) to be used in human clinical trials for disease prevention.

Base Sequence↗