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)

Two-dimensional NMR methods were used to model the possible solution structure of an intercalative complex of 9-aminoellipticine (Aell), a polycyclic pyridocarbazolamine, covalently bound to an apurinic ring-opened deoxyribose site of a duplex DNA fragment in the reduced Schiff base form. The required oligonucleotide single strand containing covalently attached aminoellipticine was obtained by reductive amination in the presence of sodium cyanoborohydride. The combined NMR-energy minimization methods were employed to refine the model structures of two distinct forms, intrahelical and extrahelical, of a control 9-mer duplex DNA, d(CGTG.dr.GTGC).d(GCACTCACG), which contains an apurinic site positioned opposite a dT residue on the complementary strand. The model structure of an aminoellipticine conjugate with the same DNA sequence, derivatized via the aforementioned covalent attachment, was also obtained by incorporating intermolecular drug-DNA and intra- and internucleotide NOE-derived proton-proton distance estimates as restraints in energy minimization routines. The indole ring system of aminoellipticine, which is inserted at the apurinic site, intercalates between and is parallel to flanking GC base pairs. The pyridinic ring of aminoellipticine, in protonated form, also stacks between cytidine and thymidine bases on the complementary strand, which is consistent with the observation that the normal sequential NOE connectivity at the 5'-C13-T14 step is broken and indeed diverted through the ellipticine moiety, e.g., C13-Aell-T14 connectivities through the Aell-H4/C5Me protons. Interestingly, the partial stacking of the pyridinic ring is observed only between the 5'-CT step vs an adjacent 5'-TC step, owing to inherently weak stacking interactions associated with the former. In the absence of any potential groups that can participate in electrostatic or hydrogen-bonding interactions with the nucleic acid, pi-pi stacking and hydrophobic contacts at the intercalation site appear to be the important factors in determining stability and conformation of the aminoellipticine-DNA conjugate. Stacking interactions in such a bistranded intercalative complexation of aminoellipticine apparently govern the formation of a single intrahelical form of a right-handed B-type DNA duplex. The overall structural features lead us to propose working models for an enzyme-like DNA cleavage activity of 9-aminoellipticine and the observed inhibition of the AP endonuclease-dependent DNA excision-repair pathway.
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PMID:High-field NMR and restrained molecular modeling studies on a DNA heteroduplex containing a modified apurinic abasic site in the form of covalently linked 9-aminoellipticine. 806 66

The interaction of the EcoRV restriction endonuclease with the dG and dC bases in its recognition sequence (GATATC) has been studied using base analogues. These modified dG and dC bases each have a single potential protein contact removed. The analogues have been incorporated into the self-complementary dodecamer d(pGACGATATCGTC) at the appropriate positions (underlined). Many of the analogues caused no change in the Tm of the duplex or else lowered the Tm by a small amount such that a duplex was still formed at temperatures suitable for enzyme assay. However, the dG analogue 2-aminopurine-1-beta-D-2'-deoxyriboside destabilized the duplex to such an extent that the 12'-mer could not be used for enzyme assays. To overcome this, a longer self-complementary 18'-mer was used with this modified base. The circular dichroism spectra of the modified base containing 12'-mers (and the 18'-mer in the case of 2-aminopurine) were very similar to the parent sequences lacking modified bases. This demonstrates the formation of B-DNA structures in all cases and similar overall conformations. The Km and kcat values for the various modified oligomers have been determined, and these data have been used to assess the roles that functional groups on the dG and dC bases play in the recognition and hydrolysis of GATATC sequences by the endonuclease. The results obtained here have been compared to the crystal structures of the EcoRV complexed with a GATATC sequence, and this has allowed a critical evaluation of the base analogue approach.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of the restriction endonuclease EcoRV with the deoxyguanosine and deoxycytidine bases in its recognition sequence. 811 Jul 83

A transposon was constructed allowing the rapid restriction mapping of plasmids. This transposon, Tn 5Map, contains a cleavage site for the I-SceI endonuclease which recognizes an 18-mer. After in vivo transposition of Tn5Map into the plasmid of interest, the plasmid is isolated and linearized with I-SceI. Splinkers labelled with digoxygenin and complementary to the left and right end of the linearized molecule are added and ligated. After partial digestion of the splinkered molecules with the restriction enzyme of interest, separation of the cleavage products in an agarose gel, and Southern transfer, the labelled fragments are visualized by the addition of the chemiluminescent substrate AMPPD and alkaline phosphatase. The restriction map can be directly read from the bottom to the top of the gel.
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PMID:Tn 5Map, a transposon for the rapid mapping of restriction sites in plasmids. 813 29

The binding of a 19-mer guanosine-rich oligodeoxyribonucleotide, TG3TG4TG4TG3T (ODN 1), to a complementary polypurine DNA target was investigated by DNase I footprinting and restriction endonuclease protection assays. Monovalent cations inhibited intermolecular purine-purine-pyrimidine triple-helical DNA formation, with K+ and Rb+ being most effective, followed by NH4+ and Na+. Li+ and Cs+ had little to no effect. Similar results were observed with the G/A-rich oligonucleotide AG3AG4AG4AG3AGCT. Kinetic studies indicated that monovalent cations interfered with oligonucleotide-duplex DNA association but did not significantly promote triplex dissociation. The observed order of monovalent cation inhibition of triplex formation is reminiscent of their effect on tetraplex formation with G/T-rich oligonucleotides. However, using electrophoretic mobility shift assays we found that the oligonucleotide ODN 1 did not appear to form a four-stranded species under conditions promoting tetraplex formation. Taken together, our data suggest that processes other than the self-association of oligonucleotides into tetraplexes might be involved in the inhibitory effect of monovalent cations on purine-pyrimidine-purine triplex formation.
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PMID:Monovalent cation effects on intermolecular purine-purine-pyrimidine triple-helix formation. 828 8

A method was investigated for monitoring the integrity of oligonucleotides in solution and in cells using fluorescence resonance energy transfer between two different fluorochromes attached to a single oligonucleotide. Ten-mer oligodeoxyribonucleotides labeled with fluorescein at one end and with rhodamine X at the other end were used. The oligomer had a specific absorption spectrum with peaks at 497 and 586 nm, which corresponded to fluorescein and rhodamine X, respectively. When excited at 494 nm, the oligomer had a specific fluorescence spectrum with peaks at 523 and 610 nm. The fluorescence intensity at 610 nm was 6-8 times higher than that at 523 nm. After digestion of the oligomer with an endonuclease, the fluorescence at 523 nm increased more than 12-15-fold but its fluorescence peak at 610 nm almost completely disappeared. To examine effects in vivo, sea urchin eggs were injected with a solution of the oligomer and excited with blue light at 470-490 nm. Two fluorescent images, a green image at 520-560 nm and a red image at above 580 nm, were obtained when a single egg was viewed under a fluorescence microscope. The ratio of the intensities of red to green fluorescence decreased in dependence on time after injection of the oligomer. These changes were not observed in eggs that had been injected with a solution of similarly double-labeled, phosphorothioate oligomer. These results indicated that unfertilized sea urchin eggs had nucleolytic activity. Analysis in vitro on supernatant of the egg homogenate indeed demonstrated the existence of nucleases. All together, our results indicate that the integrity of oligonucleotides can be estimated in living cells by monitoring the fluorescence resonance energy transfer of the double-labeled oligonucleotide.
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PMID:Detection of undegraded oligonucleotides in vivo by fluorescence resonance energy transfer. Nuclease activities in living sea urchin eggs. 855 May 91

Arbitrarily primed PCR (AP-PCR) was used to genotype 26 clinical isolates of Clostridium difficile previously analyzed by immunoblotting (IB) and 20 isolates typed by restriction endonuclease analysis (REA) with HindIII. Two levels of differentiation were achieved with the AP-PCR approach by use of two different arbitrary primers. With the 19-mer arbitrary primer T-7 (first level of differentiation), a good correlation was found between IB and AP-PCR typing. Twenty isolates grouped into six IB types were separated into seven major AP-PCR types. These seven AP-PCR groups were further discriminated into 12 subtypes after genotyping with the arbitrary primer PG-05 (second level of differentiation). The remaining six isolates, all of different IB types, showed a unique and distinct DNA banding pattern with both of the arbitrary primers, T-7 and PG-05. Twenty isolates representing 20 REA types from 15 REA groups were resolved into 13 AP-PCR DNA profiles with the arbitrary primer T-7. A good correlation was found at this level of differentiation between the major REA groups, Y and M, and AP-PCR typing. While AP-PCR with this primer failed to differentiate isolates in REA groups J, G, R, and B, AP-PCR with PG-05 resolved these four isolates into four distinct AP-PCR types. In addition, one of three M strains and one of four Y strains displayed a slightly different DNA banding pattern by AP-PCR (with PG-05) from that of the other strains in the group. We conclude that AP-PCR is a rapid and sensitive method which not only complements other typing schemes but also may be a substitute and prove to be especially suited for immediate epidemiological tracking of nosocomial infections due to C. difficile.
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PMID:Comparison of arbitrarily primed PCR with restriction endonuclease and immunoblot analyses for typing Clostridium difficile isolates. 858 95

The FokI restriction endonuclease recognizes the double-stranded (ds) 5'-GGATG-3' site and cuts at the 9th and 13th nucleotides downstream from the 5'-3' and 3'-5' strands, respectively. To elucidate the interaction between FokI and DNA, and the effect of Mg2+ on this interaction, we used FokI with various combinations of dsDNA, single-stranded (ss) DNA and oligodeoxyribonucleotides (oligos) containing a double-stranded hairpin carrying the FokI recognition site. Oligo- and dsDNA-FokI interactions showed that for fully effective recognition, two or more base-pairs were required outside the 5'-GGATG-3' site. When using FokI with ssDNA and oligos, precise cutting with no observable byproducts was observed at the 9th or 13th nucleotide. This was independent of whether the region between the recognition and cut sites was perfectly complementary or whether there were up to four mismatches in this region, or a single mismatch within the cut site. Moreover, FokI cleavage, when followed by step-wise filling-in of FokI cohesive ends in the dsDNA, allowed FokI to recleave such sites when two or more nucleotides were added, releasing 2-mer, 3-mer, or 4-mer single-stranded chains. Electrophoretic mobility shift assays showed that the DNA helix was bent when complexed with FokI (without Mg2+. Such a complex, when formed in the absence of Mg2+, did not accept the subsequently added Mg2+ for several minutes. This suggests a tight, diffusion-resistant contact between the enzyme and the cognate DNA sequence. In the presence of Mg2+, the half-life of the complex FokI and dsDNA was 12 minutes at 22 degrees C. In the absence of Mg2+, such a complex, possessing a terminally located 5'-GGATG-3' site, had a half-life of 1.5 to 2 minutes. However, if magnesium ions were present, this complex had a stability similar to that of a complex formed with dsDNA containing a centrally located 5'-GGATG-3' site.
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PMID:Structural requirements for FokI-DNA interaction and oligodeoxyribonucleotide-instructed cleavage. 863 98

Hybridisation of nucleic acid oligomers to an immobilised target has been observed in real time using evanescent field technology. A biotinylated 24-mer with random sequence including the EcoRI recognition site was immobilised via streptavidin onto a grating coupler wave guide surface. Hybridisation of 22-mer, 15-mer and 8-mer was observed. Activity of restriction endonuclease EcoRI was visualised by measurement of the loss of bound DNA after incubation.
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PMID:Label-free observation of DNA-hybridisation and endonuclease activity on a wave guide surface using a grating coupler. 865 12

One type of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), is known to increase in rat kidney DNA after the administration of a renal carcinogen, ferric nitrilotriacetate (Fe-NTA). To determine the involvement of oxygen radicals in Fe-NTA carcinogenesis, we examined whether the 8-OH-Gua repair enzymes are induced in the rat kidney after Fe-NTA administration, in addition to our analysis of the 8-OH-Gua levels in the DNA, because the 8-OH-Gua repair activity is known to be induced in mammalian cells by oxidative stress due to ionizing radiation. The 8-OH-Gua repair enzyme activity was determined with an endonuclease assay using a 22-mer double strand DNA, which contains 8-OH-Gua at a specific position. A significant increase in the 8-OH-Gua repair activity was observed in the rat kidney after a single intraperitoneal injection of Fe-NTA (p < 0.01). This is the first report on the induction of the repair activity for 8-OH-Gua after treatment with a chemical carcinogen. This assay will be useful for evaluating the carcinogenicity of oxygen radical-forming chemicals.
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PMID:Increase in the 8-hydroxyguanine repair activity in the rat kidney after the administration of a renal carcinogen, ferric nitrilotriacetate. 878 99

The renal carcinogen, ferric nitrilotriacetate (Fe-NTA), is known to induce oxidative stress and the subsequent formation of a type of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), in the rat kidney (Umemura et al., 1990). Using an improved DNA isolation method (Nakae et al., 1995), which reduces the background level of 8-OH-Gua, we found a five-fold increase in the 8-OH-Gua level in kidney DNA after a single i.p. injection of Fe-NTA. On the basis of the report that 8-OH-Gua repair activity is enhanced after cells are exposed to oxidative stress due to ionizing radiation (Bases et al., 1992), the measurement of 8-OH-Gua repair activity will also be useful to assess cellular oxidative stress. The 8-OH-Gua repair enzyme activity was determined with an endonuclease assay using a 22 mer DNA that contains 8-OH-Gua at a specific position. A five-fold increase in the 8-OH-Gua repair activity as compared with the control, was observed in the target organ, the rat kidney, 120 h after Fe-NTA administration. In the non-target organ, the liver, the increase was not as large (two-fold). This simple assay of oxidative DNA damage repair will be useful for evaluating the carcinogenicity of oxygen radical forming chemicals, in addition to chemical analyses of oxidative DNA damage.
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PMID:Increased 8-hydroxyguanine levels in DNA and its repair activity in rat kidney after administration of a renal carcinogen, ferric nitrilotriacetate. 896 57


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