PubMed Health⌕ Search

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 775 records · Page 43Linked to original sources
PubMed2003

[Construction of nucleotide excision repair gene XPB antisense RNA expression plasmid and its functions].

BACKGROUND & OBJECTIVE: Nucleotide excision repair is an important pathway for cellular DNA damage repair. The drug resistance of tumor cell is often companied with the enhanced expression of DNA repair genes. Down-regulation of DNA repair capacity by antisense strategy can increase the drug sensitivity of tumor cells. The aim of this study was to construct the eukaryotic expression plasmid pcDNA-XPB/AS (XPB: xeroderma pigmentosum B) and to investigate the function of XPB gene and its roles in chemotherapeutic drug sensitivity in lung cancer A549 cell. METHODS: The XPB cDNA (69-520 bp) fragment amplified by reverse transcription polymerase chain reaction (RT-PCR) was inserted into pcDNA3.1/His plasmid with an inverted orientation. The recombinant plasmid was transiently transfected into A549 cells. The Adriamycin-induced DNA damage was compared between the transfected and the untransfected cells by single cell gel electrophoresis assay (SCGE). The cellular sensitivity to Adriamycin of the transfected and the untransfected cells was determined by MTT assay. RESULTS: The successful construction of antisense plasmid was proved by restriction map and sequence analysis. RT-PCR results showed that the XPB mRNA expression was inhibited in transfected A549 cells. SCGE showed that the cellular damage repair ability induced by 4.0 microg/ml Adriamycin was suppressed in transfected cells. MTT assay showed the sensitivity of the transfected cells to Adriamycin was different from the untransfected cells but without statistical meaning. CONCLUSION: The antisense plasmid constructed by the authors can down-regulate the expression of XPB mRNA in the transfected cells and inhibit the cellular DNA damage repair ability, providing a basis to further study the gene function of XPB.

Base Sequence↗
PubMed2004

AP endonuclease-independent DNA base excision repair in human cells.

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.

Cells, Cultured↗
PubMed1975

Excision-repair of gamma-ray-damaged thymine in bacterial and and mammalian systems.

The selective excision of products of the 5,6-dihydroxy-dihydrothymine type (t') from gamma-irradiated or OSO4-oxidized DNA or synthetic poly[d(A-T)] was observed with crude extracts of Escherichia coli and isolated nuclei from human carcinoma HeLa S-3 and Chinese hamster ovary cells. The results with E. coli extracts allow the following conclusion: (1) The uvrA-gene product is not required for t' excision. (2) Radiation-induced strand breakage is not required for product excision. (3) Experiments with extracts of E. coli polAexl showed that the 5' in equilibrium 3' exonuclease associated with polymerase I is responsible for the removal of t'. (4) Experiments with extracts of E. coli endo I lig 4 and the ligase inhibitor nicotinamide mononucleotide showed that polynucleotide ligase accomplishes the last strand resealing step in the excision-repair of t'. Isolated nuclei from HeLa and Chinese hamster ovary cells possess the necessary enzymes for the selective excision of t' from gamma-irradiated or osmium tetroxide oxidized DNA. Approximately 25 to 35% of the products were removed from DNA within 60 min. Unspecific DNA degradation was very low. Radiation-induced strand breakage is not required for product removal.

Cell Line↗
PubMed1998

Impairment of nucleotide excision repair by apoptosis in UV-irradiated mouse cells.

We investigated the relationship between nucleotide excision repair (NER) activity and apoptosis in UV-irradiated cells. Mouse erythroleukemia (MEL) and lymphoma (GRSL) cells exhibited enhanced sensitivity to the cytotoxic effects of UV radiation compared to hamster cell lines, although normal UV-induced hprt mutation frequencies were found. Determination of UV-induced repair replication revealed a limited capacity of MEL and GRSL cells to perform NER consistent with poor removal of cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts from transcriptionally active genes during the first 8 h after UV exposure. However, both cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts appeared to be processed to almost normal level 24 h after UV treatment. In parallel, we observed that the UV-irradiated MEL and GRSL cells suffered from severe DNA fragmentation particularly 24 h after UV exposure. Taken together, these data indicate a reduced repair of UV-induced photolesions in apoptotic cells, already established at the early onset of apoptosis. To test whether inhibition of repair in cells was due to inactivation of NER or to apoptosis-induced chromatin degradation, we performed in vitro excision assays using extracts from UV-irradiated MEL cells. These experiments showed that the NER capacity during early apoptosis was intact, indicating that slow removal of UV-induced photolesions in apoptotic cells is due to substrate modification (presumably degradation of chromatin) rather than direct inhibition of factors involved in NER.

Animals↗
PubMed2006

Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced dna damage depends on functional TFIIH.

The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.

Animals↗
PubMed2005

Recombinant expression of maize nucleotide excision repair protein Rad23 in Escherichia coli.

Nucleotide excision is a highly conserved DNA repair pathway for correcting DNA lesions that cause distortion of the double helical structure. The protein heterodimer XPC-Rad23 is involved in recognition of and binding to such lesions. We have isolated full-length cDNAs encoding two different members of the maize Rad23 family. The deduced amino acid sequences of both maize orthologues show a high degree of homology to plant and animal Rad23 proteins. The cDNA encoding maize Rad23A was cloned as an in-frame C-terminal fusion of glutathione S-transferase. This chimera was expressed in Escherichia coli as a soluble protein and purified to homogeneity using glutathione-agarose followed by MonoQ column chromatography. Purified recombinant maize Rad23 protein was used to generate polyclonal antibodies that cross-react with a approximately 48-kDa protein in extracts from plant as well as mammalian cells. The purified recombinant protein and antibodies would be useful reagents to study the biochemistry of nucleotide excision repair in plants.

Amino Acid Sequence↗
PubMed1981

Excision repair and patch size in UV-irradiated bacteriophage T4.

We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

Carrier Proteins↗
PubMed2001

Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair.

While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

Animals↗
PubMed1998

Hepsulfam induced DNA adducts and its excision repair by bacterial and mammalian 3-methyladenine DNA glycosylases.

A variety of alkylated base adducts are repaired by 3-methyladenine DNA glycosylases, one of the base excision repair enzymes. In this study, we examined the DNA adducts induced by hepsulfam and determined whether alkylated base adducts can be substrates for bacterial and mammalian 3-methyladenine DNA glycosylases by electrophoresis methods. Hepsulfam, a synthetic analogue of busulfan, is known to alkylate DNA and form interstrand cross-links. The extent of DNA interstrand cross-links induced by hepsulfam and busulfan was found to be similar but significantly lower than that induced by chlorambucil, as measured by an agarose gel assay. The major monofunctional alkylation site of hepsulfam was observed at the N7 position of guanine, and not at the N3 position of adenine. Both compounds did not exhibit any sequence selective DNA alkylation patterns. The excision of hepsulfam-induced DNA adducts has been determined by treatment with homogeneous recombinant bacterial, rat and human 3-methyladenine DNA glycosylases and successive treatments by formamidopyrimidine-DNA glycosylase. The Escherichia coli alkA protein was shown to completely excise N7 guanine adducts, whereas mammalian 3-methyladenine DNA glycosylase failed to excise them. In addition, the cytotoxicity assay showed that E. coli mutant strains defective in the alkA gene or the uvrA gene were more sensitive to killing by hepsulfam than the wild type.

Alkylation↗
PubMed1989

The cloned human DNA excision repair gene ERCC-1 fails to correct xeroderma pigmentosum complementation groups A through I.

The human DNA excision repair gene ERCC-1 complements the ultraviolet light (UV) and mitomycin C (MMC) sensitivity of CHO mutants of complementation group 1. We have investigated whether ERCC-1 is the mutated gene in cell lines from xeroderma pigmentosum (XP) complementation groups A through I by analyzing the endogenous gene in XP cells and by introduction of the gene followed by repair assays. Our studies show that ERCC-1 is not deleted or grossly rearranged in representative cell lines of 9 XP groups. Furthermore, Northern blot analysis revealed correct transcription of ERCC-1 in all groups. The cloned human ERCC-1 gene was introduced into immortalized XP cells by DNA transfection (groups A, C, D, E and F). The presence of the integrated transfected sequences was verified on Southern blots and by selection for 2 dominant marker genes that flank the ERCC-1 gene on the transfected cos43-34 DNA. ERCC-1 failed to confer a normal UV survival and UV-induced unscheduled DNA synthesis (UDS) to transfected populations. In the case of the remaining XP complementation groups (B, G, H and I), nuclear microinjection was used to introduce an ERCC-1 cDNA construct driven by an SV40 promoter into primary fibroblasts. Coinjection of the SV40 large T gene and analysis of its expression served as a control for the injection. The ERCC-1 cDNA failed to induce increased levels of UDS in the microinjected fibroblasts. We infer from these experiments that ERCC-1 is not the mutated gene in the 9 XP complementation groups examined. From a similar type of experiments we conclude that ERCC-1 is not the defective gene in UV-sensitive Cockayne's syndrome cells.

Blotting, Northern↗
PubMed2004

Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae.

DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to determine whether or not AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We previously reported that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by a model DNA alkylating agent methyl methanesulfonate (MMS) and that this sensitivity can be reduced by deleting the MAG1 3-methyladenine DNA glycosylase gene. Here we report that in the absence of the AP endonucleases, deletion of two Escherichia coli endonuclease III homologs, NTG1 and NTG2, partially suppresses MMS-induced killing, which indicates that the AP lyase products are deleterious unless they are further processed by an AP endonuclease. The severe MMS sensitivity seen in AP endonuclease deficient strains can also be rescued by treatment of cells with the AP lyase inhibitor methoxyamine, which suggests that the product of AP lyase action on an AP site is indeed an extremely toxic lesion. In addition to the AP endonuclease interactions, deletion of NTG1 and NTG2 enhances the mag1 mutant sensitivity to MMS, whereas overexpression of MAG1 in either the ntg1 or ntg2 mutant severely affects cell growth. These results help to delineate alkylation base lesion flow within the BER pathway.

Alkylation↗
PubMed1998

Different DNA polymerases are involved in the short- and long-patch base excision repair in mammalian cells.

Mammalian cells possess two distinct pathways for completion of base excision repair (BER): the DNA polymerase beta (Pol beta)-dependent short-patch pathway (replacement of one nucleotide), which is the main route, and the long-patch pathway (resynthesis of 2-6 nucleotides), which is PCNA-dependent. To address the issue of how these two pathways share their role in BER the ability of Pol beta-defective mammalian cell extracts to repair a single abasic site constructed in a circular duplex plasmid molecule was tested in a standard in vitro repair reaction. Pol beta-deficient extracts were able to perform both BER pathways. However, in the case of the short-patch BER, the repair kinetics was significantly slower than with Pol beta-proficient extracts, while the efficiency of the long-patch synthesis was unaffected by the loss of Pol beta. The repair synthesis was fully dependent on PCNA for the replacement of long patches. These data give the first evidence that in cell extracts DNA polymerases other than Pol beta are specifically involved in the long-patch BER. These DNA polymerases are also able to perform short-patch BER in the absence of PCNA, although less efficiently than Pol beta. These findings lead to a novel model whereby the two BER pathways are characterized by different protein requirements, and a functional redundancy at the level of DNA polymerases provides cells with backup systems.

Animals↗
PubMed1998

Mammalian base excision repair by DNA polymerases delta and epsilon.

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol beta. Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.

Animals↗
PubMed1982

Adaptive resynthesis of O6-methylguanine-accepting protein can explain the differences between mammalian cells proficient and deficient in methyl excision repair.

Mammalian cells have been classified as proficient (Mer(+)) or deficient (Mer(-)) in methyl excision repair in terms of their cytotoxic reactions to agents that form O(6)-alkylguanine and their abilities to reactivate alkylated adenoviruses. O(6)-Methylguanine (O(6)MeGua) is considered to be a lethal, mutagenic, and carcinogenic lesion. We measured the abilities of cell extracts to transfer the methyl group from an exogenous DNA containing O(6)MeGua to acceptor protein. The constitutive level of acceptor activity was independent of the Mer phenotype and was approximately 100,000 acceptor sites per cell. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) results in a dose-dependent decrease in the acceptor activity in extracts because the rapid reaction between endogenous O(6)MeGua and acceptor protein makes the latter unavailable for further reaction. Treatment of cells with 1 muM MNNG for 15 min or 2 muM for approximately 2 min uses up >95% of the constitutive activity. However, Mer(+) cells, which are resistant to MNNG, rapidly resynthesize new acceptor protein, and the activity returns to the basal level in approximately 90 min. In Mer(-) tumor cells and Chinese hamster cells, which are sensitive to MNNG, resynthesis is not detectable in 90 min. Mer(-) simian virus 40-transformed fibroblasts, known to have an intermediate sensitivity to MNNG, have an intermediate resynthesis rate. Treatment of cells with multiple low doses of MNNG results in the enhanced production of O(6)MeGua-accepting protein in levels 2.5-fold above the constitutive values for Mer(+) tumor cells and to approximately 1.5-fold for Mer(+) fibroblasts or Mer(-) simian virus 40-transformed cells. Such treatments reduce the activities in Mer(-) tumor cells and Chinese hamster cells. We conclude: (i) estimates of O(6)MeGua in cellular DNA shortly after treatment may be seriously in error because of the rapid repair of this lesion, and (ii) the adaptive resynthesis of acceptor protein, not its constitutive level, is the important correlate of cell resistance to methylating agents.

Animals↗
PubMed1985

Radiation-induced thymine base damage and its excision repair in active and inactive chromatin of HeLa cells.

The extent of production and excision repair of 5,6-dihydroxydihydrothymine type base (t') damage was determined in transcriptionally active and inactive chromatin of HeLa cells after exposure to 6.8 MeV electrons. It was observed that not only the yield but also rate of repair of t' products was greater in the active chromatin compared to the inactive chromatin of HeLa cells. The results strongly indicate that the conformation of chromatin is an important factor in determining the sensitivity to radiation damage and accessibility to enzymes required for repair of such damage.

Chromatin↗
PubMed1997

DNA damage-dependent recruitment of nucleotide excision repair and transcription proteins to Escherichia coli inner membranes.

The entire process of nucleotide excision repair (NER) in Escherichia coli has been reconstituted in vitro from purified proteins and defined DNA substrates. However, how this system is organized in vivo in unclear. We report here the isolation and characterization of macromolecular assemblies containing NER and transcription proteins from E. coli. This ensemble consists of at least 17 proteins. They are recruited, as a consequence of DNA damage induced by UV irradiation, to the inner membrane. The UV-induced 6-4 photoproducts are also relocated to the inner membrane following UV-irradiation of the cells. This recruitment process is dependent on the uvrA, uvrC and recA gene products. These results suggest that at least part of the repair process may associate with the inner membrane and also provide insights into understanding the cellular organization of repair processes.

Adenosine Triphosphatases↗
PubMed1995

Reconstitution of human DNA repair excision nuclease in a highly defined system.

Xeroderma pigmentosum is a hereditary disease caused by defective DNA repair. Somatic cell genetics and biochemical studies with cell-free extracts indicate that at least 16 polypeptides are required to carry out the repair reaction proper, i.e. the removal of the lesion from the DNA by the dual incisions of the damaged strand. To find out if these proteins are necessary and sufficient for excision repair, they were obtained at a high level of purity in five fractions. The mixture of these five fractions reconstituted the excision nuclease (excinuclease) activity. Using the reconstituted excinuclease, we found that the excised fragment remains associated with the post-incision DNA-protein complex, suggesting that accessory proteins are needed to release the excised oligomer.

Animals↗