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Results for “Excision Repair”

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At least 217 records · Page 12Linked to original sources

PubMed1996

Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts.

Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.

Antifungal Agents↗

PubMed2003

Enhanced DNA excision repair in CCRF-CEM cells resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, quantitated using the single cell gel electrophoresis (Comet) assay.

Enhanced DNA repair activity is important for the development of cellular resistance to alkylating agents. Here, we quantitated the kinetics of DNA excision repairs initiated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in human leukemia CCRF-CEM cells. CEM cells that had been established resistant to BCNU (CEM-R) were evaluated in comparison with parental CEM cells (CEM-S). The excision repair kinetics were quantitated as the amount of DNA single strand breaks, which were generated from the incision/excision of the damaged DNA and were diminished by the rejoining of renewed DNA, using the single cell gel electrophoresis (Comet) assay. CEM-R cells were 10-fold more resistant to BCNU than CEM-S cells, and also showed cross-resistance to melphalan and cisplatin. In response to the treatment with BCNU, both CEM-S and CEM-R cells initiated an incision/excision reaction at the end of the incubation period, and completed the rejoining process within 4 hr. While CEM-S cells could not repair the damage induced by the high concentration of BCNU, CEM-R cells completed the repair process regardless of BCNU concentrations, suggesting enhanced excision repairs in CEM-R cells. The excision repair activity of CEM-R cells was increased with regard to the incision reaction and to the rate of the repair. Similar results were obtained using ultraviolet C, suggesting enhanced nucleotide excision repair in CEM-R cells. Thus, the enhanced DNA excision repairs were successfully quantitated in the resistant leukemic cell line using the Comet assay. The evaluation of the repair activity may predict the sensitivity of cancer cells to chemotherapy and provide a clue to overcome the resistance.

Antineoplastic Agents, Alkylating↗

PubMed2002

DNA base excision repair of uracil residues in reconstituted nucleosome core particles.

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.

Animals↗

PubMed1988

Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization.

In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.

DNA Repair↗

PubMed2003

Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase beta.

Base excision repair (BER) of damaged deoxyribonucleic acid (DNA) is a multistep process during which potentially lethal abasic sites temporarily exist. Repair of these lesions is greatly stimulated by heat shock protein 70 (Hsp70), which enhances strand incision and removal of the abasic sites by human apurinic-apyrimidinic endonuclease (HAP1). The resulting single-strand gaps must then be filled in. Here, we show that Hsp70 and its 48- and 43-kDa N-terminal domains greatly stimulated filling in the single-strand gaps by DNA polymerase beta, a novel finding that extends the role of Hsps in DNA repair. Incorporation of deoxyguanosine monophosphate (dGMP) to fill in single-strand gaps in DNA phagemid pBKS by DNA polymerase beta was stimulated by Hsp70. Truncated proteins derived from the C-terminus of Hsp70 as well as unrelated proteins were less effective, but proteins derived from the N-terminus of Hsp70 remained efficient stimulators of DNA polymerase beta repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase beta also was strongly stimulated by Hsp70 although not by a truncated protein from the C-terminus of Hsp70. Sealing of the repaired site in the oligonucleotide by human DNA ligase 1 was not specifically stimulated by Hsp-related proteins. Results presented here now implicate and extend the role of Hsp70 as a partner in the enzymatic repair of damaged DNA. The participation of Hsp70 jointly with base excision enzymes improves repair efficiency by mechanisms that are not yet understood.

DNA↗

PubMed1986

Excision repair of DNA in the presence of aphidicolin.

During excision repair of UV light or dimethyl sulphate (DMS)-induced damage to DNA the patch size for actively replicating KB or T98G cells is around 20 nucleotides. When confluent T98G cells or 'quiescent' KB cells are used the patch size is around 10 nucleotides. This value can be increased to around 20 nucleotides in T98G cells if a large excess of BrdUrd is included in the repair incubation medium. With 'quiescent' KB cells the patch size is not increased by excess BrdUrd. For all of these experimental conditions, when excision repair of UV or DMS damage takes place in the presence of aphidicolin, the patch size is found to be several times that found in its absence. Given the inhibitory specificity of aphidicolin for DNA polymerase alpha these results provide additional evidence that DNA polymerase alpha plays a role in the excision repair of DNA damaged by UV light or DMS. It is postulated that aphidicolin interrupts the processivity of the DNA polymerase alpha holoenzyme and allows an exonuclease to enlarge the repair site.

Aphidicolin↗

PubMed1998

Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines.

DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis. Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene. Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts. Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types. In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate. By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions. This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion. Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells. This effect was not observed in cells deleted of the DNA polymerase beta gene. These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.

Animals↗

PubMed2001

The 19S complex of the proteasome regulates nucleotide excision repair in yeast.

Previous studies suggest that the amino-terminal ubiquitin-like (ubl) domain of Rad23 protein can recruit the proteasome for a stimulatory role during nucleotide excision repair in the yeast Saccharomyces cerevisiae. In this report, we show that the 19S regulatory complex of the yeast proteasome can affect nucleotide excision repair independently of Rad23 protein. Strains with mutations in 19S regulatory subunits (but not 20S subunits) of the proteasome promote partial recovery of nucleotide excision repair in vivo in rad23 deletion mutants, but not in other nucleotide excision repair-defective strains tested. In addition, a strain that expresses a temperature-degradable ATPase subunit of the 19S regulatory complex manifests a dramatically increased rate of nucleotide excision repair in vivo. These data indicate that the 19S regulatory complex of the 26S proteasome can negatively regulate the rate of nucleotide excision repair in yeast and suggest that Rad23 protein not only recruits the 19S regulatory complex, but also can mediate functional interactions between the 19S regulatory complex and the nucleotide excision repair machinery. The 19S regulatory complex of the yeast proteasome functions in nucleotide excision repair independent of proteolysis.

Cysteine Endopeptidases↗

PubMed1983

Lack of effect of hydroxyurea on base excision repair in mammalian cells.

The effect of hydroxyurea on the initial steps of base excision repair has been examined in mammalian cells in 3 different proliferative states: i.e., quiescent cells, asynchronously growing cells undergoing multiple divisions prior to confluence; and synchronous cell populations undergoing the first cell cycle(s) after release from quiescence. Two parameters of the base excision repair pathway were examined: (1) The direct excision of 7-methylguanine from cellular DNA in the presence of increasing hydroxyurea concentrations was quantitated by high performance liquid chromatography; (2) the effects of hydroxyurea on the uracil DNA glycosylase were examined by quantitating the levels of this base excision repair enzyme in quiescent and proliferating cells. In quiescent cells, hydroxyurea at concentrations routinely used to quantitate DNA repair had no effect on the excision rates of 7-methylguanine examined over a span of 3 days; nor was there any effect on the specific activity of uracil DNA glycosylase in confluent cells. In asynchronously proliferating mammalian cells, identical hydroxyurea concentrations had no effect on the induction of the glycosylase. In synchronous growing cells HU had no effect on the temporal sequence of induction of uracil DNA glycosylase prior to DNA replication, nor on the extent of this induction. These results suggest that hydroxyurea at concentrations generally used to measure DNA repair has no effect on base excision repair.

Animals↗

PubMed1990

9-amino-ellipticine inhibits the apurinic site-dependent base excision-repair pathway.

The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base. This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E. coli through a mechanism involving apurinic sites. We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E. coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity. 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair. Other intercalating agents with similar affinities for DNA do not induce any inhibition. In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E. coli exonuclease III. The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity. 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration.

Alkaloids↗

PubMed1975

A mutant of Eshcerchia coli K-12, URT-43, with a temperature-sensitive defect at the incision step of the excision repair mechanism.

URT-43, which has a defect in excision repair, exhibits a temperature-dependent ultraviolet survival. It was shown that URT-43 requires protein synthesis but not DNA synthesis for recovery, by examining recovery in a growth medium containing chloramphenicol or nalidixic acid. The recovery of irradiated bacteriophage lambda in URT-43 took place in a medium containing nalidixic acid at 30 degrees, but not at 41 degrees, and chloramphenicol prevented this recovery. These results seem to imply that the product of the mutated gene in URT-43 is labile. URT-43 was confirmed to have a temperature-sensitive mutation at the incision step of the excision repair mechanism by examining the nick formation of parental DNA in alkaline sucrose gradients. The release of pyrimidine dimers was reinvestigated directly by one- and two-dimensional paper-chromatography and indirectly by examining the distribution of DNA molecules synthesized after irradiation. Dimers were excised into the acid-soluble fraction when growing bacteria were incubated, but were not excised when in amino acid starved bacteria. These results suggest that URT-43 is a mutant slowly excising pyrimidine dimers because the product of a mutated gene concerned with the incision step of the excision repair mechanism is unstable.

Centrifugation, Zonal↗

PubMed1975

Physico-chemical and biological study of excision-repair of UV--irradiated phiX174 RF DNA in vitro.

We have studied excision-repair of UV-irradiated phiX174 RFI DNA in vitro with UV-specific endonuclease from Micrococcus luteus (UV-endo), DNA polymerase I from Escherichia coli and DNA ligase from phage T4 infected E. coli. Excision-repair was measured a) by physico-chemical methods, i.e. by determination of the conversion of RF I DNA into RF II DNA by UV-endo and by the subsequent conversion of RF II DNA ligase, b) by biological methods i. e. by measuring the ability of the reaction product to form phages upon incubation with spheroplasts from the appropriate strains of E. coli. Using the first method, we have shown, that more than 90% of the pyrimidine dimers can be repaired in vitro; with the latter method we have shown, that the molecules which are repaired as defined by method a) have regained full biological activity. Exonuclease III was found to be not essential for excision-repair in vitro and also did not stimulate repair. From this result we conclude that UV-endo generates 3'OH endgroups, in agreement with results obtained by Hamilton et al. (1974). The usefulness of the method presented in this paper with regard to the study of excision-repair is discussed.

Chemical Phenomena↗

PubMed2005

Monte Carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. I. Model properties and predicted trends.

DNA is constantly damaged through endogenous processes and by exogenous agents, such as ionizing radiation. Base excision repair (BER) and nucleotide excision repair (NER) help maintain the stability of the genome by removing many different types of DNA damage. We present a Monte Carlo excision repair (MCER) model that simulates key steps in the short-patch and long-patch BER pathways and the NER pathway. The repair of both single and clustered damages, except double-strand breaks (DSBs), is simulated in the MCER model. Output from the model includes estimates of the probability that a cluster is repaired correctly, the fraction of the clusters converted into DSBs through the action of excision repair enzymes, the fraction of the clusters repaired with mutations, and the expected number of repair cycles needed to completely remove a clustered damage site. The quantitative implications of alternative hypotheses regarding the postulated repair mechanisms are investigated through a series of parameter sensitivity studies. These sensitivity studies are also used to help define the putative repair characteristics of clustered damage sites other than DSBs.

DNA↗

PubMed1986

Proliferation-dependent regulation of base excision repair in normal and SV-40 transformed human cells.

The effect of SV-40 viral transformation of human cells on the proliferation-dependent regulation of base excision repair was examined. Normal human diploid WI-38 fibroblasts and SV-40 transformed WI-38 cells were used to examine whether the process of transformation perturbed the regulation of base excision repair. The regulation of excision repair enzymes in each cell type was examined by quantitating: the specific activities of the base excision repair enzymes uracil DNA glycosylase and hypoxanthine DNA glycosylase as a function of cell growth; and immunological analysis of the uracil DNA glycosylase using monoclonal antibodies to human uracil DNA glycosylase. In each cell type, both DNA glycosylase activities were increased as a function of cell proliferation; the extent of enhancement being greater in the transformed cells. As defined by ELISA and immunoblot analysis, the induced uracil DNA glycosylases in both cell types share the same determinants. Although other repair processes are altered upon SV-40 transformation, these results suggest that the proliferation-dependent regulation of these base excision repair enzymes is retained in SV-40 transformed human cells.

Cell Division↗

PubMed1992

Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae.

Excision repair of DNA is an important cellular response to DNA damage caused by a broad spectrum of physical and chemical agents. We have established a cell-free system in which damage-specific DNA repair synthesis can be demonstrated in vitro with nuclear extracts from the yeast Saccharomyces cerevisiae. Repair synthesis of UV-irradiated plasmid DNA was observed in a radiation dose-dependent manner and was unaffected by mutations in the RAD1, RAD2, RAD3, RAD4, RAD10, or APN1 genes. DNA damaged with cis-platin was not recognized as a substrate for repair synthesis. Further examination of the repair synthesis observed with UV-irradiated DNA revealed that it is dependent on the presence of endonuclease III-sensitive lesions in DNA, but not pyrimidine dimers. These observations suggest that the repair synthesis observed in yeast nuclear extracts reflects base excision repair of DNA. Our data indicate that the patch size of this repair synthesis is at least seven nucleotides. This system is expected to facilitate the identification of specific gene products which participate in base excision repair in yeast.

Cell Nucleus↗

PubMed2006

Different organization of base excision repair of uracil in DNA in nuclei and mitochondria and selective upregulation of mitochondrial uracil-DNA glycosylase after oxidative stress.

Oxidative stress in the brain may cause neuro-degeneration, possibly due to DNA damage. Oxidative base lesions in DNA are mainly repaired by base excision repair (BER). The DNA glycosylases Nei-like DNA glycosylase 1 (NEIL1), Nei-like DNA glycosylase 2 (NEIL2), mitochondrial uracil-DNA glycosylase 1 (UNG1), nuclear uracil-DNA glycosylase 2 (UNG2) and endonuclease III-like 1 protein (NTH1) collectively remove most oxidized pyrimidines, while 8-oxoguanine-DNA glycosylase 1 (OGG1) removes oxidized purines. Although uracil is the main substrate of uracil-DNA glycosylases UNG1 and UNG2, these proteins also remove the oxidized cytosine derivatives isodialuric acid, alloxan and 5-hydroxyuracil. UNG1 and UNG2 have identical catalytic domain, but different N-terminal regions required for subcellular sorting. We demonstrate that mRNA for UNG1, but not UNG2, is increased after hydrogen peroxide, indicating regulatory effects of oxidative stress on mitochondrial BER. To examine the overall organization of uracil-BER in nuclei and mitochondria, we constructed cell lines expressing EYFP (enhanced yellow fluorescent protein) fused to UNG1 or UNG2. These were used to investigate the possible presence of multi-protein BER complexes in nuclei and mitochondria. Extracts from nuclei and mitochondria were both proficient in complete uracil-BER in vitro. BER assays with immunoprecipitates demonstrated that UNG2-EYFP, but not UNG1-EYFP, formed complexes that carried out complete BER. Although apurinic/apyrimidinic site endonuclease 1 (APE1) is highly enriched in nuclei relative to mitochondria, it was apparently the major AP-endonuclease required for BER in both organelles. APE2 is enriched in mitochondria, but its possible role in BER remains uncertain. These results demonstrate that nuclear and mitochondrial BER processes are differently organized. Furthermore, the upregulation of mRNA for mitochondrial UNG1 after oxidative stress indicates that it may have an important role in repair of oxidized pyrimidines.

Bacterial Proteins↗

PubMed2000

Efficient DNA base excision repair in ataxia telangiectasia cells.

Ataxia telangiectasia (A-T) cells are sensitive to a broad range of free-radical-producing and alkylating agents. Damage caused by such agents is in part repaired by base excision [base excision repair (BER)]. Two BER pathways have been demonstrated in mammalian cells: a single-nucleotide-insertion pathway and a long-patch pathway involving resynthesis of 2-10 nucleotides. Although early studies failed to detect DNA-repair defects in A-T cells exposed to ionizing radiation and radiomimetic agents, more recent experiments performed in non-dividing A-T cells and the demonstrated interaction of the A-T-mutated protein (ATM) with the BRCA1 gene product suggest that a DNA-repair defect may underlie, at least in part, the radiation sensitivity in A-T cells. We have analysed BER of a single abasic site or a single uracil in two A-T families, using an in vitro BER system. In both families, the mutation involved was homozygous and completely inactivated the ATM protein. No difference was observed between affected individuals and heterozygous or homozygous wild-type relatives in their capacity to perform DNA repair by either one-nucleotide insertion or the long-patch pathway. Hence, the putative DNA-repair defect in A-T cells, if any, does not involve BER.

Ataxia Telangiectasia↗

PubMed2003

Mode of inhibition of short-patch base excision repair by thymine glycol within clustered DNA lesions.

Clustered DNA damage, where two or more lesions are located proximally to each other, is frequently induced by ionizing radiation. Individual base lesions within a cluster are repaired by base excision repair. In this study we addressed the question of how thymine glycol (Tg) within a cluster would affect the repair of opposing lesions by human cell extracts. We have found that Tg located opposite to an abasic site does not affect cleavage of this site by apurinic/apyrimidinic (AP) endonuclease. However, Tg significantly compromised the next step of the repair. Although purified DNA polymerase beta was able to incorporate the correct nucleotide (dAMP) opposite to Tg, the rate of incorporation was reduced by 3-fold. Tg does not affect 5'-sugar phosphate removal by the 2-deoxyribose-5-phosphate (dRP) lyase activity of DNA polymerase beta, but further processing of the strand break by purified DNA ligase III was slightly diminished. In agreement with these findings, although an AP site located opposite to Tg was efficiently incised in human cell extract, only a limited amount of fully repaired product was observed, suggesting that such clustered DNA lesions may have a significantly increased lifetime in human cells compared with similar single-standing lesions.

Carbon-Oxygen Lyases↗