Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated the effect of pifithrin-alpha (PFTalpha), a chemical inhibitor of p53, on DNA double-strand break (DSB) repair in mammalian chromosomes. Thymidine kinase-deficient mouse fibroblasts were stably transfected with DNA substrates containing one or two recognition sites for yeast endonuclease I-SceI embedded within a herpes simplex virus thymidine kinase gene. Genomic DSBs were induced by introducing an I-SceI expression plasmid into cells in the presence or absence of 20 microM PFTalpha. From cells containing the DNA substrate with a single I-SceI site we recovered low-fidelity nonhomologous end-joining (NHEJ) events in which one or more nucleotides were deleted or inserted at the DSB. From cells containing the substrate with two I-SceI sites we recovered high-fidelity DNA end-joining (precise ligation (PL)) events. We found that treatment of cells with PFTalpha caused a 5-10-fold decrease in recovery of PL but decreased recovery of NHEJ by less than two-fold. Deletion sizes associated with NHEJ were unaffected by treatment with PFTalpha. Our work suggests the possibility that p53 facilitates high-fidelity DSB repair while playing little or no role in mutagenic NHEJ.
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PMID:Suppression of high-fidelity double-strand break repair in mammalian chromosomes by pifithrin-alpha, a chemical inhibitor of p53. 1250 64

The p53 tumor suppressor protein is involved in apoptosis and cell cycle checkpoints. We have shown recently that p53 also facilitates base excision repair (BER). To further examine p53 involvement in the regulation of BER we chose to focus on 3-methyladenine DNA glycosylase (3-MeAde DNA glycosylase), the first enzyme acting in the BER pathway. 3-MeAde DNA glycosylase activity was found to be modulated by the p53 protein. This modulation was dependent on the type of genotoxic stress used. Gamma-irradiation damage resulted in activation of glycosylase, which was enhanced by p53. Doxorubicin and hydrogen peroxide (H2O2) treatment, although inducing p53 stabilization, did not cause the activation of glycosylase. Nitric oxide (NO) resulted in activation of 3-MeAde DNA glycosylase. Surprisingly this activation was down regulated by wild-type p53. The down regulation of 3-MeAde DNA glycosylase activity was due to trans repression of glycosylase mRNA by p53. Furthermore, we found that AP endonuclease (APE) activity was not altered by NO. Our study provides evidence for a possible antimutagenic role for p53 following exposure of cells to NO species. In the absence of p53, NO exposure results in elevation of 3-MeAde DNA glycosylase activity that results in elevation in the number of AP sites in DNA. At the same time, APE activity does not rise and removal of the AP sites is not further processed resulting in a mutator phenotype. When p53 is present, it down regulates the transcription of 3-MeAde DNA glycosylase. This provides a new model by which p53 prevents the creation of a mutator phenotype.
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PMID:The role of p53 in base excision repair following genotoxic stress. 1455 12

Incubation of gradient purified human spermatozoa, which are routinely maintained in media prior to IVF and intracytoplasmic sperm injection (ICSI), induced DNA strand breaks (up to 89 nicks x 10(-3) bp) and chromatin release. Unlike highly dispersed Alu repeat sequences, the centromeric heterochromatin was much less susceptible to endonuclease attack. In addition to chromatin release, the permeability of the sperm membrane was altered as evidenced by reduced accessibility of sperm nuclei to decondensation factors in mouse embryo extracts. Hybridization of cDNA microarrays with DNA released from spermatozoa revealed a consistent hypersensitivity of certain genes to endogenous cleavage including TP53, VHL (tumour suppressors), BRCA1 (breast cancer), NOS1 (neurotransmitter), PECAM1, FLT1 (angiogenesis) and CDKN1C (cell cycle/imprinted). N-tert-butyl hydroxylamine (NTBH), a derivative of the anti-teratogenic alpha-phenyl-N-t-butyl nitrone (PBN) and synthetic superoxide dismutase (SOD)/catalase mimetics inhibited chromatin release and sustained or dissipated relative mitochondrial membrane potential. Together, these results show a link between the hyperactivation of sperm mitochondria and chromosomal damage of specific genes in vitro, and that the potential risk of disruption of paternally contributed genes can be circumvented by antioxidants which are known to target mitochondria.
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PMID:Gene-specific chromatin damage in human spermatozoa can be blocked by antioxidants that target mitochondria. 1465 2

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously methylated CG sites within the p53 tumor suppressor gene.
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PMID:Endogenous 5-methylcytosine protects neighboring guanines from N7 and O6-methylation and O6-pyridyloxobutylation by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. 1471 10

The role of p53 in apoptosis and the contrasting p53 status in tumors prompted us to investigate the bleomycin-induced apoptosis in p53-null human leukemia HL-60 cells (bleomycin at 160 microM for 7.5 h). Cells with apoptotic phenotype increased from 0.87% in controls to 9.40% in bleomycin-treated cells. Both the enzymes, caspase-3 and -8, were activated. Furthermore, the apoptotic phenotypes totally disappeared with zVAD-fmk, a caspase inhibitor. Besides, cytochrome c release from mitochondria happened simultaneously to apoptotic phenotypes, shrinkage of mitochondria but being independent of the mitochondrial permeability transition, since cyclosporine A and bongkrekic acid were inefficient on induced apoptosis. On the other hand, incubations with bleomycin (BLM) did not result in detectable changes in the expression of Bcl-2- and Bax-mRNA neither Bcl-2- or Bax-proteins. In conclusion, we suggest that BLM can produce apoptosis independently of p53 through three mechanisms: i) at the nuclear level by its endonuclease activities; ii) at the cell membrane, by activating caspases; and iii) at the mitochondria by releasing cytochrome c. These results indicate that BLM-induced apoptosis in HL-60 cells results from the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.
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PMID:Induction of apoptosis by bleomycin in p53-null HL-60 leukemia cells. 1471 7

Analysis of genetic changes is often hampered by insufficient starting DNA from limited clinical tissue specimens. We employed ligation-mediated PCR (LM-PCR) for global amplification of the genome to overcome this limitation, generating up to 5 microg of representative amplicons of genomic DNA from as little as one cell. We demonstrate successful global genome amplification in high-quality starting DNA source like laser-captured cultured cells, as well as partially degraded starting DNA from old formalin-fixed paraffin-embedded tissue sections. This process generates adaptor-tailed templates that can be repeatedly amplified almost ad infinitum. We have further modified this technique such that, instead of a single endonuclease digest, we can achieve higher amplicon coverage by combining 3 endonuclease digests prior to LM-PCR. As tested by examining amplification of STS sequences scattered genome-wide, the coverage was improved from the published 70% to 96%. The faithful representation of global losses and gains in the amplified genomic DNA was confirmed by array-comparative genomic hybridization. Further, we exemplify the utility of this technique for finer p53 point mutation analysis by PCR-SSCP. This technique is thus a clinically useful tool for globally amplifying and archiving DNA from finite sources like paraffin tissue sections, providing a potentially unlimited resource for genetic analyses.
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PMID:LM-PCR permits highly representative whole genome amplification of DNA isolated from small number of cells and paraffin-embedded tumor tissue sections. 1516 12

G --> T transversion mutations in the p53 tumor suppressor gene are characteristic of smoking-related lung tumors, suggesting that these genetic changes may result from exposure to tobacco carcinogens. It has been previously demonstrated that the diol epoxide metabolites of bay region polycyclic aromatic hydrocarbons present in tobacco smoke, e.g., benzo[a]pyrene diol epoxide (BPDE), preferentially bind to the most frequently mutated guanine nucleotides within p53 codons 157, 158, 248, and 273 [Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Science 274, 430-432]. However, the methodology used in that work (ligation-mediated polymerase chain reaction in combination with the UvrABC endonuclease incision assay) cannot establish the chemical structures and stereochemical identities of BPDE-guanine lesions. In the present study, we employ a stable isotope-labeling HPLC-MS/MS approach [Tretyakova, N., Matter, B., Jones, R., and Shallop, A. (2002) Biochemistry 41, 9535-9544] to analyze the formation of diastereomeric N(2)-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNA sequences. (15)N-labeled dG was placed at defined positions within DNA duplexes containing 5-methylcytosine at all physiologically methylated sites, followed by (+/-)-anti-BPDE treatment and enzymatic hydrolysis of the adducted DNA to 2'-deoxynucleosides. Capillary HPLC-ESI(+)-MS/MS was used to establish the amounts of (-)-trans-N(2)-BPDE-dG, (+)-cis-N(2)-BPDE-dG, (-)-cis-N(2)-BPDE-dG, and (+)-trans-N(2)-BPDE-dG originating from the (15)N-labeled bases. We found that all four N(2)-BPDE-dG diastereomers were formed preferentially at the methylated CG dinucleotides, including the frequently mutated p53 codons 157, 158, 245, 248, and 273. The contributions of individual diastereomers to the total adducts number at a given site varied between 70.8 and 92.9% for (+)-trans-N(2)-BPDE-dG, 5.6 and 16.7% for (-)-trans-N(2)-BPDE-dG, 2.1 and 8.5% for (-)-cis-N(2)-BPDE-dG, and 0.5 and 8.3% for (+)-cis-N(2)-BPDE-dG. The relative yields of the minor N(2)-BPDE-dG stereoisomers were elevated at the sites of inefficient adduction, while the major (+)-trans-BPDE lesion was even more dominant at the frequently adducted sites. The introduction of 5-methyl groups at adjacent cytosine bases increased the yields of N(2)-BPDE-dG diastereomers, probably a result of favorable hydrophobic interactions between BPDE and 5-methylcytosine. The targeted formation of N(2)-BPDE-dG at (Me)CG dinucleotides within the p53 gene is consistent with the high prevalence of G --> T transversions at these sites in smoking-induced lung cancer.
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PMID:Formation of diastereomeric benzo[a]pyrene diol epoxide-guanine adducts in p53 gene-derived DNA sequences. 1520 94

The tumor suppressor p53 protein stimulates nuclear base excision repair (BER) in vitro. In response to certain cellular stresses, p53 translocates to mitochondria, where it can trigger an apoptotic response. However, a potential role for p53 in modulating mitochondrial DNA repair has not yet been examined. In this study, we show that p53 also modulates mitochondrial BER. Uracil-initiated BER incorporation, which measures flux through the entire BER pathway, was lower in mitochondrial extracts from nonstressed p53 knockout mice than in wild type. The addition of recombinant p53 complemented the BER incorporation in p53 knockout extracts and stimulated BER in wt extracts. The activities of three major mitochondrial DNA glycosylases were similar in extracts from wild-type and knockout animals. Likewise, AP endonuclease activity was unaffected by the absence of p53. Gel shift experiments with recombinant p53 demonstrated that p53 did not bind to the uracil-containing substrate used in the repair assay. Polymerase gamma gap-filing activity was less efficient in p53 knockout extracts, but it was complemented with the addition of recombinant p53. Thus, we conclude that p53 may participate in mtBER by stimulating the repair synthesis incorporation step.
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PMID:p53 functions in the incorporation step in DNA base excision repair in mouse liver mitochondria. 1520 69

The tumor suppressor p53 plays an important role in the regulation of cellular response to DNA damage. Recent studies suggest that p53 is able to bind DNA with certain structural alterations in a sequence-independent manner and to interact with several molecules involved in DNA repair. This study was undertaken to test the hypothesis that p53 may participate in sensing oxidative DNA damage, the most frequently occurring spontaneous DNA lesion, and modulate its repair by the base excision repair (BER) machinery. Using synthetic DNA containing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG), we showed that p53 was pulled down together with two BER proteins, human 8-oxoguanine glycosylase (hOGG1) and AP endonuclease (APE). Functional analysis showed that p53 significantly enhanced the sequential activities of hOGG1 and APE in excising the 8-oxoG nucleotide from DNA in vitro. The ability of p53 to enhance the removal of oxidized DNA bases was further demonstrated in vivo using a pair of p53 isogenic lines. HCT116 p53+/+ cells exhibit a more rapid removal of 8-oxoG from DNA than p53-/- cells exposed to the same levels of reactive oxygen species (ROS) stress. Together, these results suggest that p53 participates in sensing oxidative DNA damage and modulates BER function in response to persistent ROS stress.
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PMID:Role of p53 in sensing oxidative DNA damage in response to reactive oxygen species-generating agents. 1534 9

Epidemiological studies have indicated that ataxia-telangiectasia (AT) heterozygotes in AT families have an increased risk of cancer, particularly of breast cancer (BC). However, in BC case-control studies, no significant differences were found in the frequency of ATM mutations between patients and controls. In such studies missense mutations were found more frequently than truncating mutations, suggesting that the cancer risk depends on mutation type. To investigate this possibility, we assessed the risk of BC according to the type and position of the ATM truncating mutation in extended AT families. DNA or RNA that had been isolated from blood or buccal cells of AT children and their relatives was screened for ATM germ-line mutations using restriction endonuclease fingerprinting, the protein truncation test, fluorescence-assisted mismatch analysis, and direct sequencing. The standardized incidence ratio of cancer associated with ATM heterozygosity status and type of mutation was estimated. We tested for genotype-phenotype correlations by simulations, permuting mutations among parental branches. No significant difference was found in the relative risk of breast cancer or any other type of cancer based on mutation type. However, the occurrence of BC may be associated with truncating mutations in certain binding domains of the ATM protein (e.g., P53/BRCA1, beta-adaptin, and FAT domains; P = 0.006). In this limited sample set, the presence of missense or truncating ATM mutations was not associated with different cancer risks. The risk of BC appeared to be associated with the alteration of binding domains rather than with the length of the predicted ATM protein.
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PMID:Cancer risk according to type and location of ATM mutation in ataxia-telangiectasia families. 1539 Jan 80


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