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 have developed a strategy by which the nature of phosphodiester bond breaks produced by various DNA-repair endonucleases and also other nucleases, can be characterized. A purified apurinic/apyrimidinic (AP) specific endonuclease from a permanently established mouse plasmacytoma cell-line (MPC-11) has been examined with respect to the exact incision site generated at the baseless site. By the aid of enzymatic treatment with calf intestinal phosphatase, the 3'-phosphatase activity of T4-polynucleotide kinase, chemical modification with piperidine in addition to the Maxam-Gilbert sequencing procedure, followed by separation on a DNA-sequencing gel, the nature of the cleaved phosphodiester bond, both 3' and 5' to the cleavage site, has been established. The AP-specific endonuclease investigated was classified as a class II AP-endonuclease according to the four possible classes of AP-endonuclease with respect to the termini produced. By use of this technique each single damaged and cleaved site can be investigated separately.
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PMID:Analysis of cleavage products of DNA repair enzymes and other nucleases. Characterization of an apurinic/apyrimidinic specific endonuclease from mouse plasmacytoma cells. 245 3

DNA photosensitization by several furocoumarins (including 3-carbethoxypsoralen (3-CPs), 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and angelicin was investigated by using DNA sequencing methodology. 3-CPs induces photo-oxidation of guanine residues leading to alkali-labile sites in DNA (revealed by hot piperidine), whereas 8-MOP, 5-MOP and angelicin do not. There is a preferential photo-oxidation of G when located on the 5' side of GG doublets, likely to reflect a better accessibility of the G moiety in such a context. Mechanisms operating via both radicals (type I) and singlet oxygen (type II) are involved in the photo-oxidation of G residues by 3-CPs. Photo-oxidized G residues are produced independently of the formation of photoadducts, and scavengers of singlet oxygen or radicals do not inhibit photobinding of 3-CPs to DNA. This leads us to propose that covalent photoadducts arise from the intercalated excited sensitizer molecules, whereas G photo-oxidations are produced either by electron transfer reactions involving bound 3-CPs or by energy transfer to molecular oxygen, thereby producing singlet oxygen that subsequently reacts with guanine bases. Quantification of both types of DNA lesions indicated that in vitro photo-oxidized G residues are produced in DNA by 3-CPs plus ultraviolet light at least to the same extent as photoadducts, under our conditions. A calf thymus redoxyendonuclease, equivalent to the endonuclease III of Escherichia coli, specific for oxidative DNA damages, recognizes and cleaves DNA at sites of photo-oxidized G residues. The extent of the cleavage by this enzyme was close to that observed by hot piperidine and followed the amount of photo-oxidized G residues produced when the lifetime of excited oxygen species is modified. The redoxyendonuclease did not incise DNA treated with 8-MOP, 5-MOP or angelicin plus ultraviolet light. The exonuclease III and endonuclease IV of E. coli also involved in the repair of oxidative DNA damage, convert the replicative form I of 3-CPs-treated DNA to replicative form II. This suggests that the lesions recognized by these enzymes are apurinic-like lesions. In view of the low toxicity and mutagenicity of 3-CPs, DNA photo-oxidation products induced by the photodynamic effect of 3-CPs are likely to be efficiently taken care of by the DNA repair system(s). It is clear that 3-CPs photo-induces several classes of DNA damage, including oxidative damage.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Oxidative DNA damage photo-induced by 3-carbethoxypsoralen and other furocoumarins. Mechanisms of photo-oxidation and recognition by repair enzymes. 247 51

Under certain circumstances sequence-specific inhibition of gene expression may be achieved in intact cells using exogenous anti-sense oligodeoxynucleotides. The efficacy of this approach to investigating gene function is limited in part by the rapid serum nuclease mediated degradation of oligodeoxynucleotides in culture media. In order to determine the relative contributions of 3'-exonuclease, 5'-exonuclease and endonuclease activity in fetal calf serum to oligodeoxynucleotide destruction, we have tested chimeric N-ras anti-sense sequence molecules protected against exonuclease attack with terminal methylphosphonate diester linkages. An 18-mer with two methylphosphonate diester linkages at the 3'-terminus, a 20-mer with two methylphosphonate diester groups at both ends, and the 16-mer 3'-methylphosphonate monoester components of their respective piperidine hydrolysates were totally resistant to venom phosphodiesterase, whereas the 16-mer 3'-hydroxyl components of the hydrolysates were rapidly degraded. Both the chimeric oligodeoxynucleotides and 3'-methylphosphonate monoesters were considerably more stable than normal 3'-hydroxyl oligodeoxynucleotides at 37 degrees C in McCoy's 5A medium containing 15% heat inactivated fetal calf serum. Typically 20-30% of the former (initial concentration 10-100 microM) remained intact at 20 h as compared to the latter which were 88-100% degraded in 4 h and undetectable at 20 h. We conclude that a 3'-phosphodiesterase activity is a predominant nuclease responsible for oligodeoxynucleotide degradation by fetal calf serum, and that for cell culture studies, significant protection of oligodeoxynucleotides may be achieved by incorporating 3'-terminal methylphosphonate diester or even monoester end groups.
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PMID:Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups. 255 58

We report the DNA sites damaged by the antitumor drug, nimustine hydrochloride (ACNU), in highly reiterated DNA sequences of rat glioma cells. A reiterated fragment of 370 base pairs (bp), obtained after Hind III restriction endonuclease digestion of rat glioma C6 or 9L cells DNA, was divided into 167 and 203 bp by subsequent Hae III enzyme reaction. The reaction of end-labelled 167 and 203 bp fragments with ACNU resulted in scission breaks corresponding to the locations of guanine. Moreover, ACNU and subsequent piperidine hydrolysis produced more frequent breaks of the phosphodiester bonds at the guanine positions, thus forming alkali-labile sites. These results indicate that the preferred site of DNA strand scission induced by ACNU is at guanine positions.
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PMID:In vitro damage of isolated DNA from two brain tumor cell lines induced by a water-soluble antitumor nitrosourea. 316 79

The DNA labile sites induced by two nitrosoureas, nimustine (ACNU) and ramustine (MCNU) synthesised in Japan, have been examined in highly reiterated DNA sequences of rat glioma cells. Reiterated fragments of 167 and 203 base pairs (bp), obtained after Hind III and Hae III restriction endonuclease digestion of rat glioma cells DNA, were used as target DNA sequences to determine the labile sites. In vitro reaction with ACNU and MCNU resulted in scission products corresponding to the locations of guanine. Subsequent piperidine hydrolysis produced more frequent breaks of the phosphodiester bonds at guanine positions, thus forming alkali-labile sites.
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PMID:DNA lability induced by nimustine and ramustine in rat glioma cells. 323 17

DNA damages caused by various anticancer nitrosourea compounds such as ACNU and MCNU were studied. Reiterated fragments of 167 and 203 base pairs (bp) were obtained after Hind III and Hae III restriction endonuclease digestion of 9L rat brain tumor DNA. The end-labeled reiterated fragments were reacted with ACNU and MCNU, which resulted in the scission breaks corresponding to the locations of guanine on an extended Maxam-Gilbert sequencing gel. Subsequent piperidine hydrolysis yielded scission products more frequently. These results indicate that nitrosoureas such as ACNU and MCNU generate DNA scission breaks and/or alkali-labile sites preferentially at the position of guanine moieties in rat brain tumor DNA.
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PMID:[Analysis of DNA damage induced by nitrosourea derivatives in rat brain tumor cells using a sequencing procedure]. 342 52

A DNA sequencing technique was applied to the highly reiterated DNA from HeLa S3 cells in order to detect DNA damage induced by the antitumor drug, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). A DNA reiterated fragment of 92 base pairs (bp) was isolated by gel electrophoresis after EcoRI and EcoRI restriction endonuclease digestion. In the defined sequence of the 92 bp fragment, ACNU caused damage and modifications primarily at guanine moieties, leading to alkali-labile sites as determined by subsequent piperidine reaction on an extended Maxam-Gilbert sequencing gel. These results indicate that guanine moieties in double-stranded DNA are preferentially vulnerable to ACNU over other base moieties.
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PMID:Detection of DNA sites damaged by 1-(4-amino-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) using a DNA sequencing procedure. 348 78

Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage. One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method. For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed. The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry. The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA. In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates. The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported.
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PMID:Synthesis and enzymatic processing of oligodeoxynucleotides containing tandem base damage. 992 34

The relationship between purified transcription factor p50 binding and ultraviolet light-induced DNA damage formation in the NF-kappa B promoter element was investigated. The effect of bound transcription factor on cyclobutane dimer formation was quantified using Maxam-Gilbert analysis of irradiated substrate digested with T4 phage endonuclease V. Two methods were employed for cleaving (6-4) photoproducts. Sites of (6-4) photoproducts cleaved by piperidine showed a general suppression in the presence of bound p50 protein similar to that observed for cyclobutane dimers. In contrast to piperidine, digestion with ultraviolet damage endonuclease (UVDE) from Saccharomyces pombe subsequent to cyclobutane dimer reversal by photolyase displayed a broader spectrum of damaged sites. Whereas some of these sites were suppressed by bound p50 protein, some remained unaffected and one site showed increased (6-4) photoproduct induction. These data illustrate the advantage of UVDE over piperidine for studying (6-4) photoproducts at the sequence level and suggest that this approach may be useful for footprinting transcription factor binding in other promoters.
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PMID:Bound transcription factor suppresses photoproduct formation in the NF-kappa B promoter. 1120 59

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


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