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:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chemical methods for the synthesis of short deoxyribooligonucleotides containing methyl and phenylphosphonodiester linkages have been developed. The interaction of two such nonionic dinucleotide analogs, T(pCH3)T and T(pC6H5)T, with several enzymes has been investigated. Because of the phosphonate linkage each dinucleotide exists as a diastereomeric pair as shown by thin layer chromatography and enzymatic studies. Both isomers of each dinucleotide can be phosphorylated by T4-polynucleotide kinase in the presence of [gamma-32P]ATP. Only one of the diastereoisomers of each dinucleotide is slowly hydrolyzed by snake venom phosphodiesterase and acts as an inhibitor of the enzyme-catalyzed hydrolysis of 5'-labeled oligothymidylic acid. Both isomers of each dinucleotide analog are completely resistant to hydrolysis by spleen phosphodiesterase.
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PMID:Synthesis and enzymatic properties of deoxyribooligonucleotides containing methyl and phenylphosphonate linkages. 22 43

Some peculiarities of activation of (ADP-ribose) polymerase by DNA fragments were studied. DNA fragments were produced by the digestion of calf thymus DNA by micrococcal nuclease and with a subsequent enzymatic modification of their end groups by nuclease S1, polynucleotide kinase of phage T4 and alkaline phosphatase. The dependence of the activating effect of DNA on the chemical structure of its end groups was established. It was shown that the terminal phosphate groups are involved in the formation of a catalytically active complex of (ADP-ribose) polymerase with DNA.
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PMID:[The role of terminal DNA groups in the activation of (ADP-ribose)polymerase]. 139 Dec 6

Two generally applicable [35S]phosphorothioate postlabeling procedures for the HPLC analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been developed based upon [32P]phosphate postlabeling assays described by Gupta and Randerath et al. In one procedure, benzo[a]pyrene (B[a]P)-modified DNA was digested to nucleoside 3'-phosphates by micrococcal nuclease and spleen phosphodiesterase and the adducted nucleotides were extracted with 1-butanol. The adducted nucleoside-3'-phosphates were 5'-thiophosphorylated by T4 polynucleotide kinase (T4PNK) and adenosine 5'-O-(3-[35S]thiotriphosphate) to yield [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts. Although thiophosphorylation of B[a]P-DNA adducts was slower than the corresponding phosphorylation reaction, similar recoveries of the postlabeled adducts were achieved with longer incubation times and higher concentrations of T4PNK. A major advantage of this procedure over the 32P-postlabeling procedure is that the resistance of phosphorothioates to degradation by phosphatases allows selective removal of the unlabeled 3'-phosphate from the [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts by brief treatment with alkaline phosphatase. [35S]B[a]P-nucleoside-5'-phosphorothioate adducts were also prepared using a nuclease P1/prostatic acid phosphatase DNA degradation method. For anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA, overall adduct recoveries were substantially higher with the nuclease P1/prostatic acid phosphatase method (48-51%) than with the micrococcal nuclease/spleen phosphodiesterase/alkaline phosphatase method (22-29%). There were no significant differences in the HPLC profiles of the [35S]phosphorothioate-postlabeled adducts obtained from these two procedures. HPLC analysis of B[a]P-DNA adducts formed in B[a]P-treated hamster embryo cell cultures demonstrated the formation of two major adducts, (+)syn-BPDE-deoxyguanosine-5'-phosphorothioate and (+)anti-BPDE-deoxyguanosine-5'-phosphorothioate, along with other minor adducts. Based upon an overall adduct recovery of 20% and 0.5 mol as the detection limit of this 35S-postlabeling/HPLC assay, the sensitivity of this assay is 1 adduct/10(8) nucleotides for a 60 micrograms DNA sample. This method offers the advantages of using 35S which has a longer half-life and lower radioactive decay energy than 32P and the ability to prepare PAH-DNA adducts at the monophosphorothioate level which greatly facilitates separation of individual 35S-postlabeled PAH-DNA adducts by HPLC.
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PMID:Detection and identification of benzo[a]pyrene-DNA adducts by [35S]phosphorothioate labeling and HPLC. 202 54

A new sensitive 32P-postlabeling assay for DNA adducts has been developed in which DNA is hydrolyzed initially by nuclease P1 and prostatic acid phosphatase instead of micrococcal nuclease and spleen phosphodiesterase as employed in previous postlabeling procedures. When DNA containing bulky adducts, X1, X2, .....Xn, is digested with nuclease P1 at pH 5, normal nucleotides are released as 5'-monophosphates, pN, while adducts are excised as 5'-phosphorylated dinucleotides, pXipN, because internucleotide linkages on the 3' side of X resist attack by nuclease P1. Addition of prostatic acid phosphatase to such a digest results in 5'-dephosphorylation of the nucleotides to normal nucleosides, N, and adducted dinucleotides, XipN, carrying a 5'-terminal free hydroxyl group. The dinucleotides but not nucleosides are converted to 5'-32P-labeled dinucleotides, [32P]pXipN, by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. Upon mapping on polyethyleneimine--cellulose anion-exchange TLC, the labeled dinucleotide adducts produce characteristic autoradiographic fingerprints. Alternatively, they are further digested with snake venom phosphodiesterase to yield 5'-monophosphates, [32P]pXi and pN. TLC profiles of the monophosphate adducts are distinct from those of the dinucleotides. These reactions provide the basis of the new 32P-postlabeling scheme, which is compared in this paper with a previously reported protocol yielding adducts in the form of 5'-32P-labeled 3',5'-bisphosphates, [32P]pXip. The results show that the availability of three different types of 32P-postlabeled derivatives for the same adduct aids in the analysis and chromatographic characterization of DNA adducts from diverse exogenous and endogenous sources.
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PMID:A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates. 254 10

Dimethyl sulfate was used to prepare 7-methyl-2'-deoxy-guanosine 3'-monophosphate (7-methyl-dGMP), which was ring-opened in alkali to 2'-deoxy-N5-methyl-N5-formyl-2,5,6-triamino-4-oxopyrimidine 3'-monophosphate (ROM-dGMP). ROM-dGMP was not dephosphorylated by nuclease P1 in contrast to normal deoxynucleotides. It was efficiently 5'-phosphorylated by T4 polynucleotide kinase. When methylated DNA was alkali-treated and digested with micrococcal nuclease, spleen phosphodiesterase and nuclease P1, ROM-dGMP was formed and this was labeled with [gamma-32P]-ATP in the presence of polynucleotide kinase. Ring-opening and P1 treatment appear methods of choice for 32P-post-labeling of 7-alkylguanines in DNA.
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PMID:Ring-opened 7-methylguanine nucleotides are resistant to nuclease P1 digestion and good substrates to polynucleotide kinase. 254 53

Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained. The purified enzyme had an Mr of 38000 +/- 3800. The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate. A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed. The polynucleotide kinase had an absolute requirement for a divalent cation. Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity. The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M. The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate. A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively. At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times. The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.
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PMID:Purification and kinetic properties of polynucleotide kinase from rat testes. 298 15

The structure of an oligodeoxyribonucleotide may be determined by a simple two-dimensional separation on a polyethyleneimine-cellulose thin layer sheet. Chromatography in the first dimension fractionates by chain length a nested set of fragments that are generated by subjecting the oligomer to partial spleen phosphodiesterase degradation and then labelling their non-common ends with 32P using polynucleotide kinase. A subsequent in situ treatment with nuclease Bal 31 produces labelled mononucleotides, and these are identified by chromatography in the second dimension. Since the method does not identify the 3' terminal nucleotide, a convenient procedure involving 3' end labelling followed by enzymatic digestion to monomers has been developed for this purpose. This approach to sequence analysis also has the advantage of permitting assignment of the identity and location of any modified or unusual bases within the oligonucleotide.
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PMID:A new method for sequence analysis of oligodeoxyribonucleotides. 298 53

Blood samples were volunteered by workers in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. DNA was isolated from peripheral white blood cells and digested with micrococcal nuclease, spleen phosphodiesterase and nuclease P1. The DNA digest was then incubated with [gamma-32P]ATP and polynucleotide kinase. Aromatic adducts present in the digest that were resistant to nuclease P1 were thus 32P-labelled while unmodified nucleotides were not. The 32P-labelled adducts were resolved by t.l.c. and detected by autoradiography. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene (high greater than 0.2, medium 0.05-0.2, low less than 0.05 microgram BP/m3 air). Aromatic adducts were found to be present in DNA from 3/4 samples from the high exposure group, 8/10 samples from the medium exposure group. 4/18 samples from the low exposure group and 1/9 samples from the unexposed controls. The levels of adducts found in the high and medium group samples ranged up to 1 adduct in 10(7) nucleotides but the levels formed in the low exposure group samples were not significantly different from those in unexposed controls. No differences related to the smoking habits of the subjects were observed. Most of the DNA adducts detected had chromatographic mobilities distinct from those formed when the 7,8-diol 9,10-oxide of BP reacted with DNA. The results indicate that highly-exposed individuals are more likely to contain aromatic DNA adducts in their white blood cells, but large interindividual variations were evident. In addition, multiple samples from the same subjects indicate that qualitative and quantitative changes in adduct patterns occur with time. This pilot study suggests that 32P-postlabelling may be useful in monitoring human exposure to known and to previously unidentified environmental genotoxic agents.
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PMID:Monitoring occupational exposure to carcinogens: detection by 32P-postlabelling of aromatic DNA adducts in white blood cells from iron foundry workers. 334 20

Polydeoxycytidylic acid (poly dC) was incubated with excess acrolein. A Nensorb 20 nucleic acid purification cartridge was used to bind the polymeric material in the poly dC/acrolein reaction mixture. The non-polymeric material eluted from this column had a UV absorbance four times higher than that of the control. The fluorescence spectrum of the eluted material did not correspond to that of unmodified cytosine. Separate aliquots of the reaction mixture were digested to deoxynucleotide 3'-monophosphates by incubation with micrococcal nuclease and spleen phosphodiesterase. The products were converted to 32P-labeled deoxynucleotide 3',5'-bisphosphates by incubation with T4 polynucleotide kinase and excess [gamma-32P]ATP. The 3'-monophosphate was selectively removed by incubation with nuclease P1. Two-dimensional thin-layer chromatography (TLC) on polyethyleneimine cellulose (PEI)-cellulose and detection of 32P-labeled deoxynucleotide 5'-monophosphates by autoradiography failed to provide evidence for the formation of an acrolein adduct of deoxycytidine 5'-monophosphate. When acrolein-modified deoxycytidine 3'-monophosphate was 32P post-labeled, a new product, which co-chromatographed with UV markers synthesized by reaction of acrolein with deoxycytidine 5'-monophosphate, was detected. These data show that acrolein-modified deoxycytidine 3'-monophosphates are substrates for 32P labeling by T4 polynucleotide kinase and are stable under the assay conditions employed. The inability to detect the acrolein-modified nucleotides after reaction with poly dC in vitro suggests that the modified bases are lost from poly dC by cleavage of the N-glycosyl bond resulting in the formation of an abasic site.
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PMID:Implications for the formation of abasic sites following modification of polydeoxycytidylic acid by acrolein in vitro. 337 Jun 25

A 32P-postlabeling method has been employed to detect the in vitro and in vivo modification of DNA by the mycotoxin sterigmatocystin (ST). ST-modified DNA was initially incubated under buffered alkaline conditions to convert unstable ST-N7-guanine moieties to stable, putative ST-formamidopyrimidine derivatives. DNA was subsequently digested with micrococcal nuclease and spleen phosphodiesterase, and the resulting ST-modified nucleotides, purified by reverse-phase thin-layer chromatography (TLC), were labeled at the 5' position via incubation with [gamma-32P]ATP and T4 polynucleotide kinase. 32P-labeled ST-nucleotides were separated by reverse-phase and anion-exchange TLC. Cerenkov quantitation of excised TLC fractions indicated that ST-DNA moieties could be detected with a sensitivity of 1 ST adduct in 3-5 X 10(7) nucleotides. Initial enzymatic digestion of ST-modified DNA was found to yield ST-modified di- and trinucleotides which, upon 32P-labeling followed by incubation with nuclease P1, liberated unmodified 5'-terminal nucleotides suggesting that ST-formamidopyrimidine-modified DNA was a poor substrate for micrococcal nuclease and spleen phosphodiesterase. Dose-dependent ST-DNA adduct formation was detected in the liver of male Fischer 344 rats over a 27-fold range of ST administered (0.33-9 mg/kg). In addition, ST-DNA adducts, formed in rats given a 9 mg/kg dose, were found to persist up to 105 days after treatment at a level of 0.5% of the 2-h value. Loss of these adducts from liver DNA was observed to exhibit a triphasic profile: rapid loss during the first 24 h (t 1/2 = 12 h) followed by a slower decline from 1 to 14 days post dosing (t 1/2 = 7 days) and an extremely slow decline from days 14 to 105 post treatment (t 1/2 = 109 days). This experimental approach to the study of mycotoxin-DNA interactions permits the quantitative description of DNA modification in ST-treated animals. Further refinement of this approach may be useful in defining the precise relationship between ST exposure and tumorigenesis in ST-exposed human populations.
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PMID:Formation and persistence of sterigmatocystin--DNA adducts in rat liver determined via 32P-postlabeling analysis. 404 87


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