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PubMed2004

Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors.

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position -3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position -6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position -19 to +8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH.

Adenosine Triphosphate↗

PubMed1998

Identification of 5'-deoxyribose phosphate lyase activity in human DNA polymerase gamma and its role in mitochondrial base excision repair in vitro.

Mitochondria have been proposed to possess base excision repair processes to correct oxidative damage to the mitochondrial genome. As the only DNA polymerase (pol) present in mitochondria, pol gamma is necessarily implicated in such processes. Therefore, we tested the ability of the catalytic subunit of human pol gamma to participate in uracil-provoked base excision repair reconstituted in vitro with purified components. Subsequent to actions of uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, human pol gamma was able to fill a single nucleotide gap in the presence of a 5' terminal deoxyribose phosphate (dRP) flap. We report here that the catalytic subunit of human pol gamma catalyzes release of the dRP residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The heat sensitivity of this activity suggests the dRP lyase function requires a three-dimensional protein structure. The dRP lyase activity does not require divalent metal ions, and the ability to trap covalent enzyme-DNA complexes with NaBH4 strongly implicates a Schiff base intermediate in a beta-elimination reaction mechanism.

Base Sequence↗

PubMed1994

Single strand and double strand DNA damage-induced reciprocal recombination in yeast. Dependence on nucleotide excision repair and RAD1 recombination.

Single strand and double strand DNA damage-induced recombination were compared in the yeast Saccharomyces cerevisiae. The non-replicating plasmid pUC18-HIS3 was damaged in vitro and introduced into yeast cells; plasmid-chromosome recombinants were selected as stable His+ transformants. Single strand damage was produced by UV irradiation at 254 nm or by psoralen photoreaction at 390 nm. Double strand damage was produced by psoralen photoreaction at 350 nm or by restriction endonuclease digestion. Recombinants were classified as resulting from gene conversion without crossing over, single plasmid integration, or multiple plasmid integration. Single and double strand DNA damage produced different patterns of recombination. In repair proficient cells double strand damage induced primarily multiple plasmid integrations, while single strand damage induced higher proportions of gene conversions and single integrations. Reciprocal recombination depended on the RAD1 gene, which is involved in both excision repair and recombination; plasmid integration induced by all forms of damage was decreased in a rad1 disruption strain. Mutation of the RAD3 excision repair gene decreased plasmid integration induced by far UV irradiation and psoralen crosslinks, but not by double strand breaks, which are not substrates of nucleotide excision repair. Double strand break-induced plasmid integration was also decreased by disruption of RAD10, which forms a complex with RAD1; disruption of RAD4 had no effect. Thus, while nucleotide excision repair genes are involved in the processing of damaged DNA to generate recombination intermediates, RAD1 and RAD10 are additionally involved in reciprocal exchange.

DNA Damage↗

PubMed1998

The DNA-dependent ATPase activity of yeast nucleotide excision repair factor 4 and its role in DNA damage recognition.

Saccharomyces cerevisiae RAD7 and RAD16 genes function together in the nucleotide excision repair of transcriptionally inactive DNA. The RAD7- and RAD16-encoded proteins exist as a tight complex named nucleotide excision repair factor 4 or NEF4. Previously, we showed that NEF4 binds UV-damaged DNA with high specificity and with a dependence upon ATP and that inclusion of NEF4 to the reconstituted nucleotide excision repair system consisting of purified NEF1, NEF2, NEF3, and replication protein A results in marked stimulation of damage-specific DNA incision. Here we show that NEF4 possesses an ATPase activity that is entirely dependent on a DNA cofactor and that double-stranded DNA is twice as effective as single-stranded DNA in activating ATP hydrolysis. Even though DNA binding is promoted by the nonhydrolyzable ATP analogue adenosine 5'-O-(thiotriphosphate) (ATPgammaS), damage binding is more proficient with ATP than with ATPgammaS. Interestingly, UV irradiation of double-stranded DNA results in a pronounced attenuation of the ATPase activity. Taken together, our results suggest a model in which ATP hydrolysis by NEF4 fuels the translocation of NEF4 on DNA in search of UV lesions and damage binding by NEF4 leads to a down-regulation of the ATPase activity. Damage-bound NEF4 could then serve as a nucleation point for the assembly of other repair components.

Adenosine Triphosphatases↗

PubMed1993

Apparent alterations in the early stage of excision repair of UV-induced DNA damages in a HeLa mutant cell line that is resistant to genotoxic stresses.

We have previously reported a cisplatin-resistant HeLa cell line featuring a cross-resistance to genotoxic stresses including ultraviolet (UV) radiation and an enhancement of plasmid reactivation. In this study, excision repair of UV-DNA adducts in this resistant cell line was investigated. Using alkaline elution analysis, this resistant cell line showed a 2-fold enhancement in damage incision-associated DNA strand breaks. Using a gel mobility shift assay, the resistant cells exhibited a 3-fold increase in nuclear proteins which specifically recognize UV-damaged DNA. However, the rate of repair synthesis in the resistant cells appeared to be the same as in their parental counterparts. Thus, recognition and incision activities, the early stage of excision repair, are altered in the resistant cells. The results suggest that phenotypic cross-resistance of this cell line to UV is probably due to an improved excision repair of UV-DNA adducts which is defective in xeroderma pigmentosum group A cells. The results are consistent with the conclusion that the early stage, including recognition and incision, of excision repair is critical in determining cellular sensitivity or resistance to DNA damage.

Cisplatin↗

PubMed2002

Human cells bearing homozygous mutations in the DNA mismatch repair genes hMLH1 or hMSH2 are fully proficient in transcription-coupled nucleotide excision repair.

The transcription-coupled nucleotide excision repair (TCNER) pathway maintains genomic stability by rapidly eliminating helix-distorting DNA adducts, such as UV-induced cyclobutane pyrimidine dimers (CPDs), specifically from the transcribed strands of active genes. DNA mismatch repair (MMR) constitutes yet another critical antimutagenic pathway that removes mispaired bases generated during semiconservative replication. It was previously reported that the human colon adenocarcinoma strains HCT116 and LoVo (bearing homozygous mutations in the MMR genes hMLH1 and hMSH2, respectively), besides manifesting hallmark phenotypes associated with defective DNA mismatch correction, are also completely deficient in TCNER of UV-induced CPDs. This revealed a direct mechanistic link between MMR and TCNER in human cells, although subsequent studies have either supported, or argued against, the validity of this important notion. Here, the ligation-mediated polymerase chain reaction was used to show at nucleotide resolution that MMR-deficient HCT116 and LoVo retain the ability to excise UV-induced CPDs much more rapidly from the transcribed vs the nontranscribed strands of active genes. Moreover, relative to DNA repair-proficient counterparts, MMR-deficient cells were not more sensitive to the cytotoxic effects of UV, and displayed equal ability to recover mRNA synthesis following UV challenge. These results conclusively demonstrate that hMLH1- and hMSH2-deficient human colon adenocarcinoma cells are fully proficient in TCNER.

Adaptor Proteins, Signal Transducing↗

PubMed1987

Direct inhibition of u.v.-induced DNA excision repair in human cells by novobiocin, coumermycin and nalidixic acid.

In permeable human fibroblasts, novobiocin, coumermycin and nalidixic acid completely inhibit u.v.-induced DNA repair synthesis, with 50% inhibition occurring at 500, 24 and 8800 microM respectively. Novobiocin also inhibits damage-specific incision of DNA in u.v.-irradiated permeable human fibroblasts by at least 75%. It has been suggested that effects of novobiocin on DNA excision repair result from changes in ATP pools; this explanation is not applicable to our data because excision repair in the permeable cell system is entirely dependent on exogenous ATP. It has also been suggested that novobiocin-induced inhibition of repair is mediated by alterations of chromatin structure recognizable by electron microscopy as gross chromatin clumping. There were no ultrastructural alterations, however, in the nuclei of permeable cells that had been incubated with 1 mM novobiocin. We conclude that, in human cells, novobiocin, coumermycin, and nalidixic acid directly inhibit the excision repair of u.v. damage to DNA, and that one locus of inhibition lies at or before the incision step. Because 1 mM novobiocin completely abolishes u.v.-induced repair synthesis in permeable cells, but inhibits damage-specific incision by only 75%, there seems to be a second site of inhibition following the incision step. The similarity between the concentrations of novobiocin, nalidixic acid and coumermycin required to inhibit u.v.-induced excision repair and the concentrations required to inhibit human DNA polymerase alpha suggest that the distal locus of inhibition may be DNA polymerase alpha-mediated repair patch synthesis. The proximal inhibitory site may be a type II DNA topoisomerase.

Aminocoumarins↗

PubMed1979

DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation.

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.

Cell Line↗

PubMed1996

Dominant negative rat DNA polymerase beta mutants interfere with base excision repair in Saccharomyces cerevisiae.

DNA polymerase beta is one of the smallest known eukaryotic DNA polymerases. This polymerase has been very well characterized in vitro, but its functional role in vivo has yet to be determined. Using a novel competition assay in Escherichia coli, we isolated two DNA polymerase beta dominant negative mutants. When we overexpressed the dominant negative mutant proteins in Saccharomyces cerevisiae, the cells became sensitive to methyl methanesulfonate. Interestingly, overexpression of the same polymerase beta mutant proteins did not confer sensitivity to UV damage, strongly suggesting that the mutant proteins interfere with the process of base excision repair but not nucleotide excision repair in S. cerevisiae. Our data implicate a role for polymerase IV, the S. cerevisiae polymerase beta homolog, in base excision repair in S. cerevisiae.

Animals↗

PubMed2005

Base-excision repair of oxidative DNA damage by DNA glycosylases.

Oxidative damage to DNA caused by free radicals and other oxidants generate base and sugar damage, strand breaks, clustered sites, tandem lesions and DNA-protein cross-links. Oxidative DNA damage is mainly repaired by base-excision repair in living cells with the involvement of DNA glycosylases in the first step and other enzymes in subsequent steps. DNA glycosylases remove modified bases from DNA, generating an apurinic/apyrimidinic (AP) site. Some of these enzymes that remove oxidatively modified DNA bases also possess AP-lyase activity to cleave DNA at AP sites. DNA glycosylases possess varying substrate specificities, and some of them exhibit cross-activity for removal of both pyrimidine- and purine-derived lesions. Most studies on substrate specificities and excision kinetics of DNA glycosylases were performed using oligonucleotides with a single modified base incorporated at a specific position. Other studies used high-molecular weight DNA containing multiple pyrimidine- and purine-derived lesions. In this case, substrate specificities and excision kinetics were found to be different from those observed with oligonucleotides. This paper reviews substrate specificities and excision kinetics of DNA glycosylases for removal of pyrimidine- and purine-derived lesions in high-molecular weight DNA.

DNA↗

PubMed2002

[Effect of benzo[a]pyrene on DNA damage and expression of genes involved in nucleotide excision repair in lung cancer cells].

OBJECTIVE: To investigate the effect of benzo[a] pyrene(BaP) on DNA damage and expression of genes involved in nucleotide excision repair[xeroderma pigmentosum group B, C, G(XPB, XPC, XPG) and excision repair cross-complementing 1 (ERCC1)] in lung cancer A549 cells. METHODS: Cell survival was measured using MIT metabolic viability assay. Single cell gel assay was applied to determine the DNA damage and repair. The level of gene expression was measured by reverse transcription-polymerase chain reaction. RESULTS: The cell survival decreased from 95.0% to 70.0% after 24 h treatment with BaP of varying concentration ranging 0.625-20.000 mumol/L. The cell survival decreased to 87.0% and 73.0% respectively after 12 h and 24 h treatment with 10 mumol/L BaP, with DNA damage gradually elevated. At 12 h after 24 h treatment, the cell survival further decreased to 59.0% and DNA damage became most serious. At 24 h after 24 h treatment, cell survival recovered to 71.0%, and damaged DNA was repaired gradually. XPB and XPC gene expression increased to 4.5-fold and 11.2-fold respectively compared with basal level at 24 h treatment or 12 h after 24 h treatment with 10 mumol/L BaP respectively. However, ERCC1 and XPG gene expression was inhibited in 24 h treatment period, then recovered gradually after treatment. CONCLUSION: Benzo[a]pyrene could lead to DNA damage and expression level change of genes involved in nucleotide excision repair in lung cancer A549 cells.

Benzo(a)pyrene↗

PubMed1998

The post-incision steps of the DNA base excision repair pathway in Escherichia coli: studies with a closed circular DNA substrate containing a single U:G base pair.

The DNA base excision repair pathway is responsible for removal of oxidative and endogenous DNA base damage in both prokaryotes and eukaryotes. This pathway involves formation of an apurinic/apyrimidinic (AP) site in the DNA, which is further processed to restore the integrity of the DNA. In Escherichia coli it has been suggested that the major mode of repair involves replacement of a single nucleotide at the AP site, based on repair synthesis studies using oligonucleotide substrates containing a unique uracil base. The mechanism of the post-incision steps of the bacterial base excision repair pathway was examined using a DNA plasmid substrate containing a single U:G base pair. Repair synthesis carried out by repair-proficient ung, recJ and xon E.coli cell extracts was analyzed by restriction endonuclease cleavage of the DNA containing the uracil lesion. It was found that replacement of the uracil base was always accompanied by replacement of several nucleotides ( approximately 15) 3' of the uracil and this process was absolutely dependent on initial removal of the uracil base by the action of uracil-DNA glycosylase. In contrast to findings with oligonucleotide substrates, replacement of just a single nucleotide at the lesion site was not detected. These results suggest that repair patch length may be substrate dependent and a re-evaluation of the post-incision steps of base excision repair is suggested.

Base Composition↗

PubMed1992

Requirement for ERCC-1 and ERCC-3 gene products in DNA excision repair in vitro. Complementation using rodent and human cell extracts.

Numerous rodent cell lines exist that have defects in nucleotide excision repair of DNA caused by alterations in genes that fall into 10 different complementation groups. The precise roles in the repair of these genes are unknown. We report here that extracts from Chinese hamster ovary cells of excision repair-defective complementation groups 1 and 3 are defective in DNA excision repair in a cell-free system. In vitro complementation can be achieved by mixing extracts from the two groups with one another. In addition, extracts from a human cell line representing xeroderma pigmentosum complementation group B could complement rodent complementation group 1 extracts, but not group 3 extracts. This is consistent with an identity of the ERCC-3 and xeroderma pigmentosum group B genes. Cellular evidence points toward a defect in the incision of damaged DNA in group 1 and 3 mutants. Since the ERCC-1 and ERCC-3 proteins are required for the in vitro reaction, it appears that both gene products are directly involved in the enzymatic incision of damaged DNA, or in preincision reactions. The experiments reported here provide the biochemical basis of an approach to analyze the function of these nucleotide excision repair proteins.

Animals↗

PubMed2003

Substrate specificities and excision kinetics of DNA glycosylases involved in base-excision repair of oxidative DNA damage.

Reactive oxygen-derived species such as free radicals are formed in living cells by normal metabolism and exogenous sources, and cause a variety of types of DNA damage such as base and sugar damage, strand breaks and DNA-protein cross-links. Living organisms possess repair systems that repair DNA damage. Oxidative DNA damage caused by free radicals and other oxidizing agents is mainly repaired by base-excision repair (BER), which involves DNA glycosylases in the first step of the repair process. These enzymes remove modified bases from DNA by hydrolyzing the glycosidic bond between the modified base and the sugar moiety, generating an apurinic/apyrimidinic (AP) site. Some also possess AP lyase activity that subsequently cleaves DNA at AP sites. Many DNA glycosylases have been discovered and isolated, and their reaction mechanisms and substrate specificities have been elucidated. Most of the known products of oxidative damage to DNA are substrates of DNA glycosylases with broad or narrow substrate specificities. Some possess cross-activity and remove both pyrimidine- and purine-derived lesions. Overlapping activities between enzymes also exist. Studies of substrate specificities have been performed using either oligodeoxynucleotides with a single modified base embedded at a specific position or damaged DNA substrates containing a multiplicity of pyrimidine- and purine-derived lesions. This paper reviews the substrate specificities and excision kinetics of DNA glycosylases that have been investigated with the use of gas chromatography/mass spectrometry and DNA substrates with multiple lesions.

Animals↗

PubMed1977

Expression of an excision repair gene in transformation of Bacillus subtilis.

DNA of Bacillus subtilis proficient in excision repair (hcr+) was introduced into Angiografinpurified competent cells of an excision repair-deficient strains UVS-1 (hcr-1). The hcr+ gene was found to affect the UV-survival curve of the cells, giving rise to a UV-resistant component. However, a considerable number of colonies of the UV-resistant component consisted of cells that were not transformed to hcr+ as judged by their sensitivity to mitomycin C (MC), UV, and by their ability to reactivate UV-irradiated M2 phages. This suggests that the hcr gene may be expressed without integration. The recA function of B. subtilis was necessary for expression of UV resistance to occur. When DNA-treated cells were selected for met+ recombinants, the UV-resistant component was again found on the UV-survival curve and about half of the colonies of the UV-resistant component consisted of Hcr- cells. This result was explained by an integration-segregation model for hcr+ and met+ genes. The effect of the hcr+ gene was seen even when DNA was added after cells were irradiated with UV, although this effect was gradually diminished by delaying the time of DNA addition. A complementation effect was found between two excision repair mutations residing in two distant loci, using hcr-114 DNA as a donor and hcr-1 cells as a recipient.

Bacillus subtilis↗

PubMed2003

Nucleotide excision repair rates in rat tissues.

We have determined and compared nucleotide excision repair capability of several rat tissues by a method based on restoration of the transformation activity of UV-irradiated pBlueScript by incubation in repair-competent protein extracts. After 3 h of incubation, plasmid DNA was isolated and used to transform competent Escherichia coli cells. Damaged plasmids showed low transformation efficiency prior to incubation in repair-competent extracts. After incubation the transformation efficiency was restored to different extents permitting calculation of the repair capacity of the extracts. Our results showed that rapidly proliferating tissues such as liver, kidney and testis showed higher nucleotide excision repair capacity than slowly proliferating tissues such as heart, muscle, lung and spleen. When liver and splenocytes were stimulated to proliferation by partial hepatectomy and mitogen stimulation, their repair capability increased in parallel with the respective proliferative rates.

Animals↗

PubMed1997

Isolation of human complexes proficient in nucleotide excision repair.

More than 20 polypeptides are required for the process of nucleotide excision repair (NER) in both human and yeast cells. This pathway of excision repair has most often been viewed as an ordered multi-step process involving steps of damage recognition, incision/excision and finally repair DNA synthesis. Here we present evidence for the existence of a complex of human NER proteins pre-assembled in the absence of damaged DNA. This multi-protein complex was initially isolated from HeLa cell extracts by affinity chromatography on a matrix containing the damage recognition protein XPA. Subsequent co-immunoprecipitation and gel filtration experiments demonstrated that a significant portion of the human NER proteins was present in the form of a high molecular weight complex and that these complexes, or repairosomes, were capable of performing all steps of NER in vitro . Consistent with studies indicating that DNA polymerasesdeltaandstraightepsiloncan both function in NER, these two polymerases are found in these repairosome complexes.

Cell Line↗

PubMed1993

Post-UV survival and mutagenesis in DNA repair-proficient and -deficient strains of Escherichia coli K-12 grown in 5-azacytidine to inhibit DNA cytosine methylation: evidence for mutagenic excision repair.

Inhibition of cytosine methylation by growth in 5-azacytidine (5-azaC), did not affect the sensitivities to DNA damage induced by exposure to ultraviolet light (UV) of Escherichia coli K-12 strains AB1157 dcm+, which is fully DNA repair-proficient, LR68 (a dcm derivative of AB1157), JC3890 dcm+ uvrB, deficient in error-free excision repair, TK702 dcm+ umuC, deficient in error-prone repair, or TK501 dcm+ uvrB umuC, which lacks both excision repair and error-prone repair. However, growth in 5-azaC increased the post-UV survival of strains AB2463 recA(Def), AB2470 recB and AB2494 lexA(Ind-), which are deficient in the induction or expression of recombination repair or error-prone repair of DNA. Spontaneous mutation frequencies were increased in strains LR68, AB2463, AB2470 and AB2494 by growth in 5-azaC, but remained unaltered in strains AB1157, JC3890, TK702 or TK501. Growth in 5-azaC significantly increased UV-induced mutation frequencies in strains AB2463 and AB2470, significantly reduced UV-induced mutation in strain JC3890, but had little effect on UV-induced mutation in the other strains. The results suggest that 5-azaC may induce a normally error-free DNA repair pathway to become error-prone and therefore genotoxic.

Azacitidine↗