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 541 records · Page 30Linked to original sources

PubMed1981

Early steps of excision repair of cyclobutane pyrimidine dimers by the Micrococcus luteus endonuclease. A three-step incision model.

The early steps of excision repair of cyclobutane pyrimidine dimers are investigated. It is demonstrated that the apurinic/apyrimidinic endonuclease associated with the Micrococcus luteus uv-specific endonuclease cleaves the phosphodiester bond on the 3' side of the deoxyribose leaving a 3' hydroxy terminus and a 5' phosphoryl terminus. This nick is not a substrate for T4 polynucleotide ligase. The 3' base-free deoxyribose terminus is not a substrate for either the polymerase or the 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I. However, the 3' terminus of the nick is converted to a substrate for DNA polymerization by the action of a 5' apurinic/apyrimidinic endonuclease. A three-step model for the incision step of excision repair of cyclobutane pyrimidine dimers is presented.

Alkaline Phosphatase↗

PubMed1992

Excision repair at individual bases of the Escherichia coli lacI gene: relation to mutation hot spots and transcription coupling activity.

To determine whether base-to-base variations in the rate of excision repair influence the distribution of mutations, we have developed a method to measure UV photoproducts at individual nucleotides in the Escherichia coli chromosome. Specific gene fragments are 3' end-labeled using a sequence-specific oligonucleotide to direct the site of labeling, and photoproducts are identified by enzymatic incision. On the nontranscribed strand of the E. coli lacI gene, the cyclobutane pyrimidine dimer frequency was 2- to 8-fold higher in chromosomal DNA than in a cloned DNA fragment. The chromosomal lesion frequency corresponded to the frequency of UV-induced mutations at mutation hot spots reported in the literature. Only 0-30% of cyclobutane dimers at various sites on this strand were excised in 20 min. In contrast, repair on the transcribed strand was 80-90% complete in 20 min. However, the transcribed strand contained an excision repair "slow spot" at the site of its single mutation hot spot: At this site, no repair occurred for the first 10 min, after which repair proceeded more slowly than typical of that strand. In an mfd strain, deficient in a factor that couples repair to transcription in cell extracts, the excision rate at individual nucleotides on the transcribed strand was minimal at most sites for at least 30 min. Wild-type E. coli's bias for producing mutations at photoproducts on the nontranscribed strand, reported to require the mfd gene, therefore appears to be due to an excision repair system specific for the transcribed strand of chromosomal DNA.

Base Sequence↗

PubMed2006

Inhibition of nucleotide excision repair (NER) by microcystin-LR in CHO-K1 cells.

Microcystin-LR (MC-LR), a potent inhibitor of PP1 and PP2A protein phosphatases, is related to tumor promotion and initiation. Although the genotoxic properties of this toxin have been extensively investigated with a variety of non-mammalian and mammalian test systems, the existing results are contradictory. Based on our previous results regarding the impact of MC-LR on the processes of DNA repair we decided to examine in greater detail its effect on the capacity of nucleotide excision repair (NER). CHO-K1 cells were pre-treated with increasing doses of MC-LR (1, 10 and 20 microg/ml) and then exposed to UV radiation (25 J/m(2)). Apoptosis was analyzed to exclude the possibility of false positive results in the comet assay. The results suggest that MC-LR targets the nucleotide excision repair mechanisms by interference with the incision/excision phase as well as the rejoining phase of NER and leads to an increased level of UV-induced cytogenetic DNA damage in CHO-K1 cells.

Animals↗

PubMed1976

Excision repair of ultraviolet-irradiated deoxyribonucleic acid in plasmolyzed cells of Escherichia coli.

A system of cells made permeable by treatment with high concentrations of surcrose (plasmolysis) has been exploited to study the excision repair of ultraviolet-irradiated deoxyribonucleic acid in Escherichia coli. It is demonstrated that adenosine 5'-triphosphate is required for incision breaks to be made in the bacterial chromosome as well as in covalently closed bacteriophage lambda deoxyribonucleic acid. After plasmolysis, uvrC mutant strains appear as defective in the incision step as the uvrA-mutated strains. This is in contrast to the situation in intact cells where uvrC mutants accumulate single-strand breaks during postirradiation incubation. These observations have led to the proposal of a model for excision repair, in which the ultraviolet-specific endonuclease, coded for by the uvrA and uvrB genes, exists in a complex with the uvrC gene product. The complex is responsible for the incision and possibly also the excision steps of repair. The dark-repair inhibitors acriflavine and caffeine are both shown to interfere with the action of the adenosine 5'-triphosphate-dependent enzyme.

Acriflavine↗

PubMed2006

Malondialdehyde, a major endogenous lipid peroxidation product, sensitizes human cells to UV- and BPDE-induced killing and mutagenesis through inhibition of nucleotide excision repair.

Aldehydes are ubiquitous contaminants in the human environment. Intracellular aldehydes are mainly derived from the metabolism of polyunsaturated fatty acids and from lipid peroxidation, which is significantly elevated under oxidative stress conditions. Oxidative stress has long been suspected to be involved in many disease processes, including carcinogenesis, neurodegeneration and aging, but its mechanisms are largely unknown. Aldehydes are reactive not only toward nucleic acids but also to many amino acids, and these aldehyde-protein interactions have been suspected of affecting many cellular functions, including DNA repair. To test this possibility we determined the effect of malondialdehyde (MDA), one of the most abundant intracellular aldehyde, on ultraviolet (UV) light- and benzo(a)pyrene diol epoxide (BPDE)-induced cytotoxicity and mutagenesis in human cells. We found that MDA treatment greatly sensitized cells to both UV- and BPDE-induced cell killing and that, MDA pre-treatment significantly enhanced UV-induced mutagenesis. Using in vitro DNA repair synthesis and host cell reactivation assays we found that MDA treatment of cells greatly inhibited nucleotide excision repair for both and UV light- and BPDE-induced DNA damage. Further experiments raise the possibility that the inhibitory effect on nucleotide excision repair is mainly caused by the direct interaction of MDA with cellular repair proteins. Together these results strongly suggest that intracellular aldehydes play an important role in oxidative stress-related mutagenesis and carcinogenesis through their inhibitory effect on DNA repair mechanisms as well as on induction of DNA damage.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1988

Alternative pathways for the in vivo repair of O6-alkylguanine and O4-alkylthymine in Escherichia coli: the adaptive response and nucleotide excision repair.

The in vivo removal of three different O-alkylated bases from DNA was measured in Escherichia coli. Using monoclonal antibodies specific for O6-methylguanine, O6-ethylguanine and O4-ethylthymine we have monitored the removal of these lesions from six different strains to assess the relative contributions of the adaptive response and of nucleotide excision repair. During the first hour after DNA alkylation, O6-methylguanine, O6-ethylguanine and O4-ethylthymine lesions were repaired almost exclusively by nucleotide excision, except when the adaptive response was being constitutively expressed. In wild-type E. coli the adaptive response began to contribute to O6-methylguanine repair about one hour after alkylation, the time required for the full induction of the ada DNA methyltransferase. In contrast, the adaptive response did not play such a large role in the repair of O6-ethylguanine and O4-ethylthymine in wild-type E. coli, presumably because DNA ethylation damage is a poor inducer of the adaptive response; possible reasons for this poor induction are discussed. The repair of all three O-alkylated lesions was virtually absent in ada- uvr- bacteria suggesting that no alternative pathway is available for their repair, at least during the first two hours after alkylation. When the repair of O-alkylated bases was compromised by an ada- or by a uvr- mutation, the bacteria became more sensitive to alkylation induced killing and mutation.

Alkylation↗

PubMed1998

Involvement of molecular chaperonins in nucleotide excision repair. Dnak leads to increased thermal stability of UvrA, catalytic UvrB loading, enhanced repair, and increased UV resistance.

UvrA is one of the key Escherichia coli proteins involved in removing DNA damage during the process of nucleotide excision repair. The relatively low concentrations (nanomolar) of the protein in the normal cells raise the potential questions about its stability in vivo under both normal and stress conditions. In vitro, UvrA at low concentrations is shown to be stabilized to heat inactivation by E. coli molecular chaperones DnaK or the combination of DnaK, DnaJ, and GrpE. These chaperone proteins allow sub-nanomolar concentrations of UvrA to load UvrB through >10 cycles of incision. Guanidine hydrochloride-denatured UvrA was reactivated by DnaK, DnaJ, and GrpE to as much as 50% of the native protein activity. Co-immunoprecipitation assays showed that DnaK bound denatured UvrA in the absence of ATP. UV survival studies of a DnaK-deficient strain indicated an 80-fold increased sensitivity to 100 J/m2 of ultraviolet light (254 nm) as compared with an isogenic wild-type strain. Global repair analysis indicated a reduction in the extent of pyrimidine dimer and 6-4 photoproduct removal in the DnaK-deficient cells. These results suggest that molecular chaperonins participate in nucleotide excision repair by maintaining repair proteins in their properly folded state.

Adenosine Triphosphatases↗

PubMed1998

Stereoselectivity of human nucleotide excision repair promoted by defective hybridization.

To assess helical parameters that dictate fast or slow removal of carcinogen-DNA adducts, we probed human nucleotide excision repair (NER) activity with DNA containing L-deoxyriboses. Unlike natural lesions such as pyrimidine dimers or base adducts, L-deoxyribonucleosides (the mirror images of normal D-deoxyribonucleosides) involve neither the addition nor the loss of covalent bonds or functional groups and hence exclude modulation of repair efficiency by adduct chemistry and size. Previous studies showed that single L-deoxyribonucleosides distort DNA backbones but are accommodated in the double helix with intact hydrogen bonding between complementary strands. Here, we found that such single L-enantiomers are rejected as excision repair substrates in a NER-proficient cell extract. However, the same L-deoxyribose moiety stimulates NER activity upon incorporation into a nonhybridizing site of one or, more effectively, two base mismatches. In contrast to single L-deoxyriboses, multiple consecutive L-deoxyriboses interfere with normal hybridization; in this case, the intrinsic derangement of base pairing was sufficient to promote the excision of a cluster of three adjacent L-deoxyribonucleosides without any requirement for mismatches. Thus, using stereoselective substrates, we demonstrate the participation of a recognition subunit that guides human NER activity to sites of defective Watson-Crick strand pairing. This conformational sensor detects labile hydrogen bonds irrespective of the type of deoxyribonucleotide modification.

Base Pair Mismatch↗

PubMed1982

Cell cycle-dependent regulation of excision repair of UV damage.

The incision step of excision repair of UV-damaged DNA, represented by the accumulation of DNA strand breaks in the presence of hydroxyurea and 1-beta-D-arabinofuranosylcytosine, was measured in cultured cells of Microtus agrestis during quiescence and at different times after release from quiescence. The UV dose-dependent kinetics of enzymic incision change rapidly through the first cell cycle after release, with a maximum potential for enzymic incision and a minimum affinity of the incision system for damage sites occurring in late G1. The potential for incision falls to a lower level as cells approach a randomly proliferating state, whilst the affinity rises. Incision activity declines rapidly after UV irradiation, leaving many damage sites unrepaired; the enhanced survival seen in cells held in quiescence after irradiation is thus partly due to some other repair mode.

Animals↗

PubMed2006

XPA protein as a limiting factor for nucleotide excision repair and UV sensitivity in human cells.

Nucleotide excision repair (NER) acts on a variety of DNA lesions, including damage induced by many chemotherapeutic drugs. Cancer therapy with such drugs might be improved by reducing the NER capacity of tumors. It is not known, however to what extent any individual NER protein is rate-limiting for any step of the repair reaction. We studied sensitivity to UV radiation and repair of DNA damage with regard to XPA, one of the core factors in the NER incision complex. About 150,000-200,000 molecules of XPA protein are present in NER proficient human cell lines, and no XPA protein in the XP-A cell line XP12RO. Transfected XP12RO cell lines expressing 50,000 or more XPA molecules/cell showed UV resistance similar to normal cells. Suppression of XPA protein to approximately 10,000 molecules/cell in a Tet-regulatable system modestly but significantly increased sensitivity to UV irradiation. No removal of cyclobutane pyrimidine dimers was detected in the SV40 immortalized cell lines tested. Repair proficient WI38-VA fibroblasts and transfected XP-A cells expressing 150,000 molecules of XPA/cell removed (6-4) photoproducts from the genome with a half-life of 1h. Cells in which XPA protein was reduced to about 10,000 molecules/cell removed (6-4) photoproducts more slowly, with a half-life of 3h. A reduced rate of repair of (6-4) photoproducts thus results in increased cellular sensitivity towards UV irradiation. These data indicate that XPA levels must be reduced to <10% of that present in a normal cell to render XPA a limiting factor for NER and consequent cellular sensitivity. To inhibit NER, it may be more effective to interfere with XPA protein function, rather than reducing XPA protein levels.

Antineoplastic Agents↗

PubMed2003

Mammalian DNA base excision repair proteins: their interactions and role in repair of oxidative DNA damage.

The DNA base excision repair (BER) is a ubiquitous mechanism for removing damage from the genome induced by spontaneous chemical reaction, reactive oxygen species (ROS) and also DNA damage induced by a variety of environmental genotoxicants. DNA repair is essential for maintaining genomic integrity. As we learn more about BER, a more complex mechanism emerges which supersedes the classical, simple pathway requiring only four enzymatic reactions. The key to understand the complete BER process is to elucidate how multiple proteins interact with one another in a coordinated process under specific physiological conditions.

Animals↗

PubMed2003

UV wavelength-dependent regulation of transcription-coupled nucleotide excision repair in p53-deficient human cells.

Nucleotide excision repair (NER) prevents skin cancer by eliminating highly genotoxic cyclobutane pyrimidine dimers (CPDs) induced in DNA by the UVB component of sunlight. NER consists of two distinct but overlapping subpathways, i.e., global NER, which removes CPD from the genome overall, and transcription-coupled NER (TCNER), which removes CPD uniquely from the transcribed strand of active genes. Previous investigations have clearly established that the p53 tumor suppressor plays a crucial role in the NER process. Here we used the ligation-mediated PCR technique to demonstrate, at nucleotide resolution along two chromosomal genes in human cells, that the requirement for functional p53 in TCNER, but not in global NER, depends on incident UV wavelength. Indeed, relative to an isogenic p53 wild-type counterpart, p53-deficient human lymphoblastoid strains were shown to remove CPD significantly less efficiently along both the transcribed and nontranscribed strands of the c-jun and hprt loci after exposure to polychromatic UVB (290-320 nm). However, in contrast, after irradiation with 254-nm UV, p53 deficiency engendered less efficient CPD repair only along the nontranscribed strands of these target genes. The revelation of this intriguing wavelength-dependent phenomenon reconciles an apparent conflict between previous studies which used either UVB or 254-nm UV to claim, respectively, that p53 is required for, or plays no role whatsoever in, TCNER of CPD. Furthermore, our finding highlights a major caveat in experimental photobiology by providing a prominent example where the extensively used "nonsolar" model mutagen 254-nm UV does not accurately replicate the effects of environmentally relevant UVB.

Base Sequence↗

PubMed2001

Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair.

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.

Animals↗

PubMed2002

Defective nucleotide excision repair in yeast hpr1 and tho2 mutants.

Nucleotide excision repair (NER) and transcription are intimately related. First, TFIIH has a dual role in transcription initiation and NER and, secondly, transcription leads to more efficient repair of damage present in transcribed sequences. It is thought that elongating RNAPII, stalled at a DNA lesion, is used for the loading of the NER machinery in a process termed transcription-coupled repair (TCR). Non-transcribed regions are repaired by the so-called global genome repair (GGR). We have previously defined a number of yeast genes, whose deletions confer transcription-dependent hyper-recombination phenotypes. As these mutations cause impairment of transcription elongation we have assayed whether they also affect DNA repair. We show that null mutations of the HPR1 and THO2 genes, encoding two prominent proteins of the THO complex, increase UV sensitivity of yeast cells lacking GGR. Consistent with this result, molecular analyses of DNA repair of the RPB2 transcribed strand using T4 endo V show that hpr1 and tho2 do indeed impair TCR. However, this effect is not confined to TCR alone because the mutants are slightly affected in GGR. These results indicate that THO affects both transcription and NER. We discuss different alternatives to explain the effect of the THO complex on DNA repair.

Blotting, Northern↗

PubMed2005

Base excision repair intermediates are mutagenic in mammalian cells.

Base excision repair (BER) is the main pathway for repair of DNA damage in mammalian cells. This pathway leads to the formation of DNA repair intermediates which, if still unsolved, cause cell lethality and mutagenesis. To characterize mutations induced by BER intermediates in mammalian cells, an SV-40 derived shuttle vector was constructed carrying a site-specific lesion within the recognition sequence of a restriction endonuclease. The mutation spectra of abasic (AP) sites, 5'-deoxyribose-5-phosphate (5'dRp) and 3'-[2,3-didehydro-2,3-dideoxy-ribose] (3'ddR5p) single-strand breaks (ssb) in mammalian cells was analysed by RFLP/PCR and mutation frequency was estimated by quantitative PCR. Point mutations were the predominant events occurring at all BER intermediates. The AP site-induced mutation spectrum supports evidence for the 'A-rule' and is also consistent with the use of the 5' neighbouring base to instruct nucleotide incorporation (5'-rule). Preferential adenine insertion was also observed after in vivo replication of 5'dRp or 3'ddR5p ssb. We provide original evidence that not only the abasic site but also its derivatives 'faceless' BER intermediates are mutagenic, with a similar mutation frequency, in mammalian cells. Our findings support the hypothesis that unattended BER intermediates could be a constant threat for genome integrity as well as a spontaneous source of mutations.

Animals↗

PubMed2007

Tfb5 interacts with Tfb2 and facilitates nucleotide excision repair in yeast.

TFIIH is indispensable for nucleotide excision repair (NER) and RNA polymerase II transcription. Its tenth subunit was recently discovered in yeast as Tfb5. Unlike other TFIIH subunits, Tfb5 is not essential for cell survival. We have analyzed the role of Tfb5 in NER. NER was deficient in the tfb5 deletion mutant cell extracts, and was specifically complemented by purified Tfb5 protein. In contrast to the extreme ultraviolet (UV) sensitivity of rad14 mutant cells that lack any NER activity, tfb5 deletion mutant cells were moderately sensitive to UV radiation, resembling that of the tfb1-101 mutant cells in which TFIIH activity is compromised but not eliminated. Thus, Tfb5 protein directly participates in NER and is an accessory NER protein that stimulates the repair to the proficient level. Lacking a DNA binding activity, Tfb5 was found to interact with the core TFIIH subunit Tfb2, but not with other NER proteins. The Tfb5-Tfb2 interaction was correlated with the cellular NER function of Tfb5, supporting the functional importance of this interaction. Our results led to a model in which Tfb5 acts as an architectural stabilizer conferring structural rigidity to the core TFIIH such that the complex is maintained in its functional architecture.

Amino Acid Sequence↗

PubMedDate not supplied

Evidence for in the appearance of mRNAs of nucleotide excision repair genes, in human ovarian cancer tissues.

Nucleotide excision repair (NER) is the DNA repair pathway through which cisplatin-DNA intrastrand adduct is repaired. Clinical studies have shown that increased mRNA expression of selected genes involved in the rate-limiting step of NER, appear to be closely associated with clinical resistance to platinum agents. These specific studies have led to the possibility of an assessment of the order in which, the appearance of mRNAs of selected NER genes may occur. Included in this assessment are ERCC1, XPB, CSB, and XPA, studied in 28 ovarian cancer tumor tissue specimens. The study of these four genes, in pairs, from 28 ovarian cancer specimens, results in 168 separate observations. Based on the mRNA expression patterns of these genes in these tissues, it is suggested that ERCC1 mRNA may appear before the mRNA of any of the other genes, in an obligate fashion. This is followed by XPB mRNA; which appears before the mRNA of XPA; which in turn, may appear before CSB. This pattern is consistent with what we have reported previously, in non-malignant human bone marrow specimens from a cohort of 52 patients.

Adenocarcinoma↗

PubMed1996

Multiple involvement of nucleotide excision repair enzymes: clinical manifestations of molecular intricacies.

Nucleotide excision repair (NER) is a process required to remove DNA damage inflicted upon our skin by the short-wave bands of natural sunlight. Defective NER may result in a high risk of UV-induced skin tumors, since it occurs in patients with the inherited disorder xeroderma pigmentosum (XP). However, Cockayne's syndrome (CS) and PIBIDS (a photosensitive form of trichothiodystrophy) are also disorders with defective NER, but show no evidence of an elevated risk of cancer. In addition, many of CS and PIBIDS symptoms are difficult to explain on the basis of an NER defect only. Recent new insights into the molecular mechanisms of NER have shown additional involvements of many NER enzymes in other cellular processes. These multiple functions are likely to be the basis of the complex symptomatology of XP, CS and PIBIDS. Specific gene-targeted mouse models will probably help to solve these intricacies.

Animals↗