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)

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify. Therefore, we use the NNK analogue 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentration-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA. Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine. In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation. Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248. We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV. These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA. The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks. These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds.
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PMID:Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA. 1167 38

CYP2A6 (cytochrome P450 2A6), which was first identified as the human coumarin 7-hydroxylase, is the most important enzyme in nicotine C-oxidation. The enzyme also metabolically activates the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vitro. Polymorphisms in the CYP2A6 gene may thus impact on both smoking behavior and lung cancer susceptibility. Several different genotyping methods have been reported with conflicting results in the frequencies of CYP2A6 polymorphic variants. Thus we decided to perform a sequence analysis of the entire CYP2A6 gene. Sequencing confirmed the published CYP2A6 cDNA sequence. However, intron sequences differed considerably from the reported sequence of the CYP2A6*3 (v2) variant. Our analyses revealed that parts of introns shared homologies with the published sequence of CYP2A13. Based on our sequence data we developed a one step protocol for specific amplification of exon 3 of CYP2A6. The resulting PCR product can be used directly for restriction endonuclease digestion with XcmI and DdeI to determine the frequencies of the reported variant alleles CYP2A6*2 and CYP2A6*3. In a population of 305 African-Americans and 145 Caucasians, we found allele frequencies of 0.003 (2/610) for CYP2A6*2 and 0 (0/610) for CYP2A6*3 in African-Americans and allele frequencies of 0.014 (4/290) and 0 (0/290) in Caucasians. We conclude that both alleles are considerably less frequent in populations than previously reported.
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PMID:Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP genotyping method. 1168 23

The mutagenicity of a prominent tobacco carcinogen, benzo[a]pyrene (B[a]P), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (N(2)-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR (LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within codons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, we performed a direct quantitative analysis of N(2)-BPDE-dG originating from specific guanine nucleobases within p53- and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. (15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, the two genes most frequently mutated in smoking-induced lung cancer. (15)N-labeled DNA was annealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N(2)-BPDE-dG lesions. The extent of adduct formation at (15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of (15)N-N(2)-BPDE-dG and N(2)-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and p53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N(2)-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, an endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N(2)-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G --> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N(2)-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N(2)-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.
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PMID:Formation of benzo[a]pyrene diol epoxide-DNA adducts at specific guanines within K-ras and p53 gene sequences: stable isotope-labeling mass spectrometry approach. 1213 76

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

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

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions that arise from the loss of normal bases. APE1, the major AP endonuclease of human cells, plays a central role in the repair of AP sites through both its endonuclease and phosphodiesterase activities. A common APE1 polymorphism, a T-->G transversion (Asp 148 Glu), was previously shown to be associated with risk of lung cancer, an association that was modified by cigarette smoking. To explore the association between APE1 genotype, smoking and bladder cancer risk, we examined data from an existing case-control study of bladder cancer patients (n = 239) and control individuals (n = 215) recruited from urology clinics at 2 hospitals in North Carolina. Genotype at the polymorphic site was determined using allele-specific primer extension reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found no overall association between APE1 genotype and bladder cancer risk. In stratified analyses, however, a positive association with risk was observed with an increasing number of Glu alleles among never smokers, but not among smokers (p-value for interaction = 0.005). We can speculate that small allelic differences that are apparent in never smokers are obscured by the large amount of DNA damage found in smokers. Given the lack of established biological mechanisms, and suboptimal numbers of subjects in some exposure categories, our findings should be interpreted cautiously.
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PMID:APE1 genotype and risk of bladder cancer: evidence for effect modification by smoking. 1642 70

Oxidative DNA damage and DNA repair may mediate several cellular processes, like replication and transcription, mutagenesis and apoptosis and thus may be important for the organism development as well as its pathogenesis, including cancer. Activity of DNA repair enzymes can depend on many factors, such as gene polymorphism, mRNA and protein level, as well as enzymes activation and inhibition. Modulation of base excision repair pathway eliminating from DNA oxidatively formed lesions may be caused by the diet, inflammation and neoplastic transformation. Reactive oxygen species and some diet components induce transcription of several Base Excision Repair enzymes, e.g. major human AP-endonuclease, (APE1) and 8-oxoG-DNA glycosylase (OGG1). The carcinogenic process in human lung decreases repair activity for 8-oxoGin transcription independent manner, but increases repair activity of epsilon A and epsilon C, as measured in tumors and unchanged lung tissues of lung cancer patients. Thus, modulation of repair enzymes activities may be a cell response on their way to differentiation ot neoplastic transformation.
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PMID:Modulation of oxidative DNA damage repair by the diet, inflammation and neoplastic transformation. 1722 95

Oxidative DNA damage and DNA repair mediate the development of several human pathologies, including cancer. The major pathway for oxidative DNA damage repair is base excision repair (BER). Functional assays performed in blood leukocytes of cancer patients and matched controls show that specific BER pathways are decreased in cancer patients, and may be risk factors. These include 8-oxoguanine (8-oxoG) repair in lung and head and neck cancer patients and repair of lipid peroxidation (LPO) induced 1,N(6)-ethenoadenine (epsilonA) in lung cancer patients. Decrease of excision of LPO-induced DNA damage, epsilonA and 3,N(4)-ethenocytosine (epsilonC) was observed in blood leukocytes of patients developing lung adenocarcinoma, specific histological type of cancer related to inflammation and healing of scars. BER proteins activity depends on gene polymorphism, interactions between BER system partners and post-translational modifications. Polymorphisms of DNA glycosylases may change their enzymatic activities, and some polymorphisms increase the risk of inflammation-related cancers, colorectal, lung and other types. Polymorphisms of BER platform protein, XRCC1 are connected with increased risk of tobacco-related cancers. BER efficiency may also be changed by reactive oxygen species and some diet components, which induce transcription of several glycosylases as well as a major human AP-endonuclease, APE1. BER is also changed in tumors in comparison to unaffected surrounding tissues, and this change may be due to transcription stimulation, post-translational modification of BER enzymes as well as protein-protein interactions. Modulation of BER enzymes activities may be, then, an important factor determining the risk of cancer and also may participate in cancer development.
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PMID:Base excision repair modulation as a risk factor for human cancers. 1762 57

Cancer chemotherapy and radiotherapy kill cancer cells by inducing DNA damage, unless the lesions are repaired by intrinsic repair pathways. DNA double-strand breaks (DSB) are the most deleterious type of damage caused by cancer therapy. Homologous recombination (HR) is one of the major repair pathways for DSB and is thus a potential target of cancer therapy. Cells with a defect in HR have been shown to be sensitive to a variety of DNA-damaging agents, particularly interstrand crosslink (ICL)-inducing agents such as mitomycin C and cisplatin. These findings have recently been applied to clinical studies of cancer therapy. ERCC1, a structure-specific endonuclease involved in nucleotide excision repair (NER) and HR, confers resistance to cisplatin. Patients with ERCC1-negative non-small-cell lung cancer were shown to benefit from adjuvant cisplatin-based chemotherapy. Imatinib, an inhibitor of the c-Abl kinase, has been investigated as a sensitizer in DNA-damaging therapy, because c-Abl activates Rad51, which plays a key role in HR. Furthermore, proteins involved in HR have been shown to repair DNA damage induced by a variety of other chemotherapeutic agents, including camptothecin and gemcitabine. These findings highlight the importance of HR machinery in cancer therapy.
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PMID:Clinical relevance of the homologous recombination machinery in cancer therapy. 1795 11

RNAi is widely applied to inhibit expression of specific genes, but it is limited by variable efficiency and specificity of empirically designed siRNA or shRNA constructs. This complicates studies targeting individual genes and significantly impairs large-scale screens using genome-wide knockdown libraries. Here, we show that ectopic expression of the RISC slicer Argonaute-2 (Ago2, eIF2C2) dramatically enhances RNAi specifically for mRNA targets with perfectly matched binding sites. This effect depends on its endonuclease activity and is uncoupled from its regulation of microRNA expression. To model the application of Ago2 coexpression with shRNA knockdown, we targeted the EGF receptor (EGFR) in lung cancer cells exhibiting oncogene addiction to EGFR. Whereas multiple empirically designed shRNA constructs exhibited highly divergent efficiencies in mediating EGFR knockdown and cell killing, coexpression of Ago2 resulted in uniform and highly specific target gene suppression and apoptosis in EGFR-dependent cells. Codelivery of Ago2 with shRNA constructs or siRNA duplexes thus provides a strategy to enhance the efficacy and the specificity of RNAi in experimental and potentially therapeutic settings.
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PMID:Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites. 1859 65


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