SEARCH · PubMed Health

Results for “Excision Repair”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources

PubMed1985

Incorporation of biotin-labeled deoxyuridine triphosphate into DNA during excision repair and electron microscopic visualization of repair patches.

Biotin-labeled deoxyuridine triphosphate (BiodUTP) has the potential to be a useful affinity probe for studies on DNA repair, if it can be incorporated into DNA repair patches and does not inhibit subsequent steps in the excision repair pathway. We have synthesized BiodUTP by an improved procedure and have used permeable normal human fibroblasts to determine the effect of substituting BiodUTP for thymidine triphosphate on several steps in the excision repair pathway: incision, polymerization, ligation, and nucleosome rearrangement. The results demonstrate that BiodUTP is efficiently incorporated into repair patches and has little or no effect on the repair process. The presence of BiodUMP in ligated repair patches has been used to visualize the repair patches by electron microscopy following incubation with ferritin-labeled avidin. This approach has been used to estimate the maximum size of repair patches induced by ultraviolet radiation.

Biotin↗

PubMed1999

Cell cycle checkpoint abrogator UCN-01 inhibits DNA repair: association with attenuation of the interaction of XPA and ERCC1 nucleotide excision repair proteins.

UCN-01, an anticancer agent currently in Phase I clinical trials, has been found to potentiate the cytotoxicity of cisplatin (CDDP). Because mammalian cells remove CDDP-induced DNA adducts through the nucleotide excision repair (NER) pathway, we determined the effects of UCN-01 on NER by measuring its effects on the interaction of the repair factors XPA and ERCC1 and the phosphorylation/dephosphorylation of the repair proteins. The repair activity, as measured by an in vitro repair synthesis assay and an in vivo host-cell reactivation assay using A549 cells, was significantly reduced. Although expression of XPA and ERCC1 proteins was elevated in cells exposed to UCN-01, the treatment resulted in a decreased ERCC1 level in the Triton X-100-insoluble fraction of cell lysates. A pull-down assay using the MBP-XPA fusion protein showed a significant reduction in the binding of ERCC1 to XPA in nuclear extracts from UCN-01-treated cells compared with untreated cells, suggesting that UCN-01 reduced the XPA-ERCC1 interaction. Consistent with these data, lower repair incision activity was found in the cell extracts from UCN-01-treated cells. In vitro phosphorylation revealed that UCN-01 had no effect on the phosphorylation/dephosphorylation status of either XPA or ERCC1; however, UCN-01 caused dephosphorylation of an unidentified XPA-bound protein with an apparent molecular mass of 52 kDa. Taken together, these data demonstrate the NER-inhibitory action of UCN-01, which is associated with the inhibition of the XPA-ERCC1 interaction by UCN-01 and with the effect of UCN-01 on the phosphorylation/dephosphorylation of an XPA-bound, 52-kDa protein, the identity of which remains to be determined.

Alkaloids↗

PubMed1985

Analysis of the excision repair of nondimer DNA damage induced by solar ultraviolet radiation in ICR 2A frog cells.

The excision repair of solar uv-induced nondimer DNA damage was examined in ICR 2A frog cells through the use of the bromodeoxyuridine (BrdUrd) photolysis assay. A relatively pure population of nondimer DNA photoproducts was induced by irradiation of ICR 2A cells with the Mylar-filtered solar ultraviolet (uv) wavelengths produced by a fluorescent sunlamp followed by exposure to photoreactivating light (PRL) which removes most of the small yield of pyrimidine dimers induced by this treatment. Cultures of cells were also exposed to 254 nm uv, which induces primarily dimers, and 60Co gamma rays. Through use of a modification of the BrdUrd photolysis assay possessing enhanced sensitivity, it was found that the solar uv-induced nondimer DNA damage was repaired by a short patch repair mechanism in which less than approximately 20 nucleotides are inserted into a repaired region. Similar results were also obtained for gamma-irradiated cells. In contrast, excision repair of 254-nm-induced dimers was accomplished by a long-patch process in which an average of about 180 nucleotides are inserted into the repaired sites.

Animals↗

PubMed2003

Poly(ADP-ribose) polymerase in base excision repair: always engaged, but not essential for DNA damage processing.

Poly(ADP-ribose) polymerase (PARP-1) is an abundant nuclear protein with a high affinity for single- and double-strand DNA breaks. Its binding to strand breaks promotes catalysis of the covalent modification of nuclear proteins with poly(ADP-ribose) synthesised from NAD(+). PARP-1-knockout cells are extremely sensitive to alkylating agents, suggesting the involvement of PARP-1 in base excision repair; however, its role remains unclear. We investigated the dependence of base excision repair pathways on PARP-1 and NAD(+) using whole cell extracts derived from normal and PARP-1 deficient mouse cells and DNA substrates containing abasic sites. In normal extracts the rate of repair was highly dependent on NAD(+). We found that in the absence of NAD(+) repair was slowed down 4-6-fold after incision of the abasic site. We also established that in extracts from PARP-1 deficient mouse cells, repair of both regular and reduced abasic sites was increased with respect to normal extracts and was NAD(+)-independent, suggesting that in both short- and long-patch BER PARP-1 slows down, rather than stimulates, the repair reaction. Our data support the proposal that PARP-1 does not play a major role in catalysis of DNA damage processing via either base excision repair pathway.

Animals↗

PubMed2000

DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair.

DNA damage formation and repair are tightly linked to protein-DNA interactions in chromatin. We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication ( ARS1 ). The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides. Photoreactivation was generally faster than NER at all sites. Site-specific heterogeneity of repair was observed for both pathways. This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed. The results indicate distinct inter-actions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication.

DNA Damage↗

PubMed1999

Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision repair, whereas cells compromised for pRb function are defective only in global repair.

After exposure to DNA-damaging agents, the p53 tumor suppressor protects against neoplastic transformation by inducing growth arrest and apoptosis. A series of investigations has also demonstrated that, in UV-exposed cells, p53 regulates the removal of DNA photoproducts from the genome overall (global nucleotide excision repair), but does not participate in an overlapping pathway that removes damage specifically from the transcribed strand of active genes (transcription-coupled nucleotide excision repair). Here, the highly sensitive ligation-mediated PCR was employed to quantify, at nucleotide resolution, the repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in genetically p53-deficient Li-Fraumeni skin fibroblasts, as well as in human lung fibroblasts expressing the human papillomavirus (HPV) E6 oncoprotein that functionally inactivates p53. Lung fibroblasts expressing the HPV E7 gene product, which similarly inactivates the retinoblastoma tumor-suppressor protein (pRb), were also investigated. pRb acts downstream of p53 to mediate G(1) arrest, but has no demonstrated role in DNA repair. Relative to normal cells, HPV E6-expressing lung fibroblasts and Li-Fraumeni skin fibroblasts each manifested defective CPD repair along both the transcribed and nontranscribed strands of the p53 and/or c-jun loci. HPV E7-expressing lung fibroblasts also exhibited reduced CPD removal, but only along the nontranscribed strand. Our results provide striking evidence that transcription-coupled repair, in addition to global repair, are p53-dependent in UV-exposed human fibroblasts. Moreover, the observed DNA-repair defect in HPV E7-expressing cells reveals a function for this oncoprotein in HPV-mediated carcinogenesis, and may suggest a role for pRb in global nucleotide excision repair.

DNA Repair↗

PubMed1998

Functional analysis of human replication protein A in nucleotide excision repair.

Human replication protein A (RPA) is a three-subunit protein complex (70-, 34-, and 11-kDa subunits) involved in DNA replication, repair, and recombination. Both the 70- (p70) and 34-kDa (p34) subunits interact with Xeroderma pigmentosum group A complementing protein (XPA), a key protein involved in nucleotide excision repair. Our deletion analysis indicated that no particular domain(s) of RPA p70 was essential for its interaction with XPA, whereas 33 amino acids from the C terminus of p34 (p34Delta33C) were necessary for the XPA interaction. Furthermore, mutant RPA lacking the p34 C terminus failed to interact with XPA, suggesting that p34, not p70, is primarily responsible for the interaction of RPA with XPA. RPA stimulated the interaction of XPA with UV-damaged DNA through an RPA-XPA complex on damaged DNA sites because (i) the RPA mutant lacking the C terminus of p34 failed to stimulate an XPA-DNA interaction, and (ii) the ssDNA binding domain of RPA (amino acids 296-458) was necessary for the stimulation of the XPA-DNA interaction. Two separate domains of p70, a single-stranded DNA binding domain and a zinc-finger domain, were necessary for RPA function in nucleotide excision repair. The mutant RPA (RPA:p34Delta33C), which lacks its stimulatory effect on the XPA-DNA interaction, also poorly supported nucleotide excision repair, suggesting that the XPA-RPA interaction on damaged DNA is necessary for DNA repair activity.

Animals↗

PubMed1986

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. IV. Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair.

A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments. The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined. Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out. It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells. In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication. The REV2 mediated mutagenic process could be separated from the repair function.

DNA Repair↗

PubMed1985

Excision repair in u.v. (254 nm) damaged non-dividing human skin fibroblasts: a major biological role for DNA polymerase alpha.

We have used the eukaryotic DNA polymerase alpha inhibitor, aphidicolin, and the polymerase beta inhibitor, dideoxythymidine, to examine the role of these enzymes in excision repair of ultraviolet (u.v., 254 nm) damage induced in non-dividing (arrested) human skin fibroblasts. The effects of these drugs on u.v.-treated cells have been monitored using a simple and reproducible repair synthesis assay in parallel with viability measurements to determine the degree of inhibition of repair of potentially lethal damage. In agreement with previous studies using density gradients, repair synthesis induced by low fluences of u.v. (less than 3 J m-2) is relatively insensitive to inhibition by aphidicolin compared to high fluences where approximately 85 per cent inhibition is observed at the highest (20 micrograms/ml) aphidicolin concentration employed. However, repair of potentially lethal damage is inhibited by at least 90 per cent over the entire fluence range. Although dideoxythymidine led to considerable inhibition of repair synthesis, the result is probably an artifact under these in vivo conditions. The polymerase beta inhibitor was not toxic to u.v.-treated cells nor did it add to the toxicity of aphidicolin when the drugs were used in combination. We conclude that if the beta polymerase is involved in excision repair then its temporary (4 h) inhibition by dideoxythymidine is entirely reversible. In contrast, polymerase alpha appears to be an enzyme essential to the majority of biologically effective excision repair over the entire u.v. fluence range tested.

Aphidicolin↗

PubMed2006

Conserved XPB core structure and motifs for DNA unwinding: implications for pathway selection of transcription or excision repair.

The human xeroderma pigmentosum group B (XPB) helicase is essential for transcription, nucleotide excision repair, and TFIIH functional assembly. Here, we determined crystal structures of an Archaeoglobus fulgidus XPB homolog (AfXPB) that characterize two RecA-like XPB helicase domains and discover a DNA damage recognition domain (DRD), a unique RED motif, a flexible thumb motif (ThM), and implied conformational changes within a conserved functional core. RED motif mutations dramatically reduce helicase activity, and the DRD and ThM, which flank the RED motif, appear structurally as well as functionally analogous to the MutS mismatch recognition and DNA polymerase thumb domains. Substrate specificity is altered by DNA damage, such that AfXPB unwinds dsDNA with 3' extensions, but not blunt-ended dsDNA, unless it contains a lesion, as shown for CPD or (6-4) photoproducts. Together, these results provide an unexpected mechanism of DNA unwinding with implications for XPB damage verification in nucleotide excision repair.

Amino Acid Motifs↗

PubMed1994

Nickel(II) interferes with the incision step in nucleotide excision repair in mammalian cells.

Nickel compounds are carcinogenic to humans and experimental animals. However, the mechanisms leading to tumor formation are still not understood since the mutagenic potential is rather weak. In contrast, nickel(II) enhances the cytotoxicity and genotoxicity in combination with several other DNA-damaging agents. To elucidate possible interactions with DNA repair processes, the effect of nickel(II) on the nucleotide excision repair pathway has been investigated after UV irradiation in HeLa cells. Nickel(II) blocks the removal of cyclobutane pyrimidine dimers as determined by T4 endonuclease V-sensitive sites. When the alkaline unwinding technique was applied, significantly less transient DNA strand breaks after UV irradiation were detected in the presence of nickel(II) compared to UV alone, suggesting an inhibition of the incision step of nucleotide excision repair. Once incisions are made, the ligation of repair patches is delayed as well in nickel-treated cells, as observed by the alkaline unwinding and nucleoid sedimentation techniques. This inhibition of DNA repair is partly reversible by the addition of magnesium(II), indicating that the competition between Ni2+ and Mg2+ may provide an important mechanism for the disturbance of DNA-protein interactions involved in the repair process. Since the repair inhibition is observed at noncytotoxic concentrations of nickel(II), it may well be relevant for its carcinogenic action.

Colony-Forming Units Assay↗

PubMed2001

Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome.

In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair. A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell.

DNA Repair↗

PubMed1983

The effects of excision repair and the plasmid pKM101 on the induction of his+ revertants by chemical agents in Salmonella typhimurium.

Expansion of the Ames Salmonella/microsome mutagenesis test to include plasmid pKM101-bearing, excision repair-proficient derivatives permits 1) the identification of mutagens that require both factors for activity; 2) the identification of genotoxins through the enhancement of survival by excision repair; and 3) the classification of substances according to the effects of excision repair on their mutagenesis. Class I includes substances that require excision repair to effect mutagenesis. Class II contains substances whose mutagenesis is not affected by excision repair. Class III mutagens cause premutational lesions in DNA which are readily removed by excision repair. This classification scheme is suggested as a preliminary step in making a risk estimation for a mutagen.

4-Nitroquinoline-1-oxide↗

PubMed1991

Characterization and expression of RAD4 gene involved in nucleotide excision repair of UV-damaged Saccharomyces cerevisiae.

Saccharomyces cerevisiae express RAD4 gene for nucleotide excision repair of UV-induced DNA damages. Upon complementation with rad4-4 mutant, a 7.6 kb clone containing the RAD4 gene designated as pPC1 was isolated from a yeast genomic library. The pPC1 was further narrowed to 2.5 kb flanked with BglII and BamHI sites. The cloned RAD4 gene was found to propagate in E. coli without loss of its complementing activity. Pulse-field gel electrophoresis indicated that the cloned RAD4 gene was localized in the right arm of chromosome V. DNA-tRNA hybridization revealed that the cloned gene did not contain a suppressor tRNA gene. The rad4 mutants with various plasmids containing the cloned RAD4 gene, regardless of their copy number, had enhanced resistance against UV damages equivalent to that found in wild type. As determined by S1 nuclease digestion, the RAD4 transcript was found to be 2.3 kb in size and the S1 nuclease mapping revealed the production of a protected fragment of 760 nucleotides within the transcript. Transcriptional start point was found at 48 base pairs upstream from the first ATG codon of the translation initiation codon. The overexpressed Rad4 protein was estimated to be 89 kD and confirmed the expected size based on the actual length of RAD4 gene. Upon stationary phase culturing, E. coli cells transformed with the cloned RAD4 gene had a delayed entrance into exponential growth phase and produced reduced amount of host proteins. These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplication in E. coli.

Cloning, Molecular↗

PubMed2006

Fluorodeoxyuridine modulates cellular expression of the DNA base excision repair enzyme uracil-DNA glycosylase.

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) continues to play a pivotal role in the treatment of cancer. A downstream event of thymidylate synthase inhibition involves the induction of a self-defeating base excision repair process. With the depletion of TTP pools, there is also an increase in dUMP. Metabolism of dUMP to the triphosphate dUTP results in elevated pools of this atypical precursor for DNA synthesis. Under these conditions, there is a destructive cycle of dUMP incorporation into DNA, removal of uracil by the base excision repair enzyme uracil-DNA glycosylase (UDG), and reincorporation of dUMP during the synthesis phase of DNA repair. The end point is DNA strand breaks and loss of DNA integrity, which contributes to cell death. Evidence presented here indicates that both the nuclear and the mitochondrial isoforms of UDG are modulated by FdUrd (and 5-FU) treatment in certain cell lines but not in others. Modulation occurs at the transcriptional and post-translational levels. Under normal conditions, nUDG protein appears in G(1) and is degraded during the S to G(2) phase transition. The present study provides evidence that, in certain cell lines, FdUrd mediates an atypical turnover of nUDG. Additional data indicate that, for cell lines that do not down-regulate nUDG, small interfering RNA-mediated knockdown of nUDG significantly increases resistance to the cytotoxic effects of FdUrd. Results from these studies show that nUDG is an additional determinant in FdUrd-mediated cytotoxicity and bolster the notion that the self-defeating base excision repair pathway, instigated by elevated dUTP (FdUTP) pools, contributes to the cytotoxic consequences of 5-FU chemotherapy.

DNA Repair↗

PubMed1999

Initiation of base excision repair: glycosylase mechanisms and structures.

The base excision repair pathway is an organism's primary defense against mutations induced by oxidative, alkylating, and other DNA-damaging agents. This pathway is initiated by DNA glycosylases that excise the damaged base by cleavage of the glycosidic bond between the base and the DNA sugar-phosphate backbone. A subset of glycosylases has an associated apurinic/apyrimidinic (AP) lyase activity that further processes the AP site to generate cleavage of the DNA phosphate backbone. Chemical mechanisms that are supported by biochemical and structural data have been proposed for several glycosylases and glycosylase/AP lyases. This review focuses on the chemical mechanisms of catalysis in the context of recent structural information, with emphasis on the catalytic residues and the active site conformations of several cocrystal structures of glycosylases with their substrate DNAs. Common structural motifs for DNA binding and damage specificity as well as conservation of acidic residues and amino groups for catalysis are discussed.

Catalysis↗

PubMed1989

Physical association of base excision repair enzymes with parental and replicating DNA in BHK-21 cells.

The physical association of mammalian excision repair enzymes with DNA was examined as a function of cell proliferation. The molecular weight distribution of two nuclear base excision repair enzymes, the uracil DNA glycosylase and the hypoxanthine DNA glycosylase, were examined by sucrose step gradient analysis. The sedimentation of DNA polymerase activity as well as the distribution of parental and replicating DNA were determined simultaneously. In confluent BHK-21 fibroblasts, basal levels of both DNA glycosylases, DNA polymerase beta, and parental DNA sedimented to the 20%/40% sucrose border. In proliferating BHK-21 cells, induced levels of both DNA glycosylases, DNA polymerase alpha, and replicating DNA sedimented to the 40%/50% sucrose border. The physical association of the repair enzymes with DNA was demonstrated by detergent treatment and by DNase digestion. As defined by [35S] methionine pulse labeling analysis, newly synthesized DNA repair enzymes were localized with either parental or replicating DNA. These results suggested that the physical association of mammalian DNA repair enzymes with nuclear DNA was dependent on the proliferative state of the cell.

Cell Division↗

PubMed2001

The p53-regulated cyclin-dependent kinase inhibitor, p21 (cip1, waf1, sdi1), is not required for global genomic and transcription-coupled nucleotide excision repair of UV-induced DNA photoproducts.

The p53 tumor suppressor gene is a transcriptional activator involved in cell cycle regulation, apoptosis, and DNA repair. We have shown that p53 is required for efficient nucleotide excision repair of UV-induced DNA photoproducts from global genomic DNA but has no effect on transcription-coupled repair. In order to evaluate whether p53 influences repair indirectly through cell cycle arrest following DNA damage or plays a direct role, we examined repair in vivo in human cells genetically altered to disrupt or regulate the function of p53 and p21. Both primary human fibroblasts and HCT116 colon carcinoma cells wild type for p53 but in which the p21 gene was inactivated through targeted homologous recombination showed no decrease in global repair of UV photoproducts. Human bladder carcinoma cells mutant for p53 and containing a tetracycline-regulated p21 cDNA showed no significant enhancement of repair upon induction of p21 expression. All of the cell lines, including the mismatch repair-deficient, MLH1 mutant HCT116 cells, were proficient for transcription-coupled repair. Clonogenic survival of HCT116 cells following UV irradiation showed no dependence on p21. Therefore, our results indicate that p53-dependent nucleotide excision repair does not require the function of the p21 gene product and is independent of p53-regulated cell cycle checkpoints.

Cell Line↗