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 examined the DNA-protein interactions involved in the recognition of a specific hexameric sequence, GAATTC, by the EcoRI modification methylase by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues 2-aminopurine, 5-bromocytosine, 5-bromouracil, 2,6-diaminopurine, hypoxanthine, 5-methylcytosine, N6-methyladenine, and uracil were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the ability of the enzyme to methylate the modified substrates were monitored by determining the steady state kinetic values of the reaction in the presence of the cosubstrate, S-adenosylmethionine. The substitutions resulted in effects ranging from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those analogues that were active, whereas the octanucleotides containing hypoxanthine at the guanine site, N6-methyladenine at the first or 2-aminopurine at the second adenine site, or uracil at the second thymine site were completely inactive. The results are discussed in terms of the possible interactions between the methylase and its recognition sequence. In addition, the interactions are compared to those of the EcoRI restriction endonuclease, which has been similarly tested with the same analogue oligonucleotides. The results of that study are reported in an accompanying paper. Although both enzymes recognize the same sequence, they do so in different ways.
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PMID:The effects of base analogue substitutions on the methylation by the EcoRI modification methylase of octadeoxyribonucleotides containing modified EcoRI recognition sequences. 301 81

The construction and use of a series of positive-selection vectors are described. These plasmids encode EcoRI endonuclease, the synthesis of which is under the control of the lacUV5 promoter. The pKG2 plasmid encodes a wild-type EcoRI endonuclease. In the absence of EcoRI methylase, the endonuclease is lethal. Cloning into any of the unique restriction sites within the endonuclease-coding gene allows survival of the transformed EcoRI-methylase-less host. The pKGW and pKGS plasmids encode an altered EcoRI endonuclease which, when repressed in a lacIQ host, allows survival in the absence of the methylase. Induction with IPTG, however, results in cell death as a result of high-level EcoRI synthesis. Cloning into any of the unique restriction sites within the EcoRI gene of pKGW or pKGS allows survival of derepressed transformed cells. These vectors strongly select for cloning events which inactivate the endonuclease gene.
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PMID:Positive-selection vectors utilizing lethality of the EcoRI endonuclease. 301 39

DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.
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PMID:Cloning the DdeI restriction-modification system using a two-step method. 302 41

Specific IgG antibodies were raised in rabbits against purified EcoRI methylase and restriction endonuclease. Post embedding labeling experiments, using the protein A-gold technique, were made with paraformaldehyde-glutaraldehyde fixed cells, embedded in Lowicryl K4M resin at low temperatures. Labeling with methylase-specific antibodies showed 60-70% of gold particles in the cytoplasm and 30-40% at the cell envelope, whereas the use of restriction enzyme-specific antibodies led to a distribution of 10-30% in the cytoplasm and 70-90% in the cell envelope. The results coincide with the proposed function of the enzymes: in the cytoplasm methylase protects the cells' own DNA from self-destruction, and the restriction endonuclease cuts foreign DNA when entering the cell.
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PMID:In situ distribution of EcoRI methylase and restriction endonuclease in cells of Escherichia coli Bs 5. 303 75

Human SY5Y neuroblastoma cells which were differentiated in culture by treatment with 7S murine nerve growth factor for 5 weeks and selection with aphidicolin (L. Jensen, Dev. Biol. 120:56-64, 1987) demonstrated a considerably slower rate of removal of DNA adducts of benzo[a]pyrene, benzo[a]pyrenediolepoxide, and N7-methylguanine than did undifferentiated mitotic cells. A dramatic decline in unscheduled DNA synthesis induced by UV radiation was similarly observed. DNA polymerase beta and uracil DNA glycosylase were unchanged after differentiation, DNA polymerase alpha and DNA methylase decreased roughly threefold, and total apurinic-apyrimidinic endonuclease activity increased roughly threefold after treatment.
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PMID:A reduced rate of bulky DNA adduct removal is coincident with differentiation of human neuroblastoma cells induced by nerve growth factor. 314 94

In order to detect possible m5C photoproducts, highly purified rat liver DNA-cytosine methyltransferase was used to specifically generate m5C with a radioactive methyl group. When these DNAs were subjected to a large dose (10 kJ/m2) of 254 nm or 302 nm ultraviolet light (UVB) to enhance the yield, two labeled photoproducts were detected and isolated by reverse phase HPLC after formic acid hydrolysis. Further studies using acetone as a triplet state sensitizer and UVB irradiation suggested that photoproduct II was activated via a triplet state while the more polar photoproduct I was not. Photoreversion of the purified photoproducts with 10 kJ/m2 254 nm light demonstrated the following reactions: Photoproduct I regenerated m5C, while photoproduct II is split and regenerated m5C and photoproduct I. These results suggest that photoproduct I is monomeric while photoproduct II dimeric, and from the latter's elution position possibly a cyclobutyl type dimer arising from a reaction with an adjacent cytosine. Using d[TTG] and d[Cm5CG] as models of typical sequences, irradiation with 10 kJ/m2 254 nm or 302 nm, respectively, gave rise to a small component having altered mobility in sequencing gels. The altered mobility trinucleotides were resistant to degradation by PI and micrococcal nucleases as expected from photodimerization of the pyrimidine bases. Furthermore, oligonucleotide substrates containing m5C were synthesized and shown to be susceptible to T4 endonuclease v action at locations consistent with d[Cm5C] photodimer formation when irradiated in the UVB range.
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PMID:UV-induced photoproducts of 5-methylcytosine in a DNA sequence context. 337 57

A DNA methyltransferase was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1. The enzyme recognized the sequence GATC and methylated deoxyadenosine solely in GATC sequences. Host DNA, which contains GATC sequences, but not PBCV-1 DNA, which contains GmATC sequences, was a good substrate for the enzyme in vitro. The DNA methyltransferase activity was first detected about 1 h after viral infection; PBCV-1 DNA synthesis and host DNA degradation also began at about this time. The appearance of the DNA methyltransferase activity required de novo protein synthesis, and the enzyme was probably virus encoded. Methylation of DNAs with the PBCV-1-induced methyltransferase conferred resistance of the DNAs to a PBCV-1-induced restriction endonuclease enzyme described previously (Y. Xia, D. E. Burbank, L. Uher, D. Rabussay, and J. L. Van Etten, Mol. Cell. Biol. 6:1430-1439). We propose that the PBCV-1-induced methyltransferase protects viral DNA from the PBCV-1-induced restriction endonuclease and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.
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PMID:DNA methyltransferase induced by PBCV-1 virus infection of a Chlorella-like green alga. 353 3

The effect of pyrimidine photodimers on transmethylation reactions catalyzed by a highly purified rat liver DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) that exhibits maintenance and de novo methylation activities was studied in vitro, using the viral substrates M13 mp9 replicative form (RF) DNA and the hemimethylated analog formed from primed synthesis of phage DNA in the presence of 2'-deoxy-5-methylcytidine 5'-triphosphate. These DNAs were irradiated with UVB (280-340 nm) at 900-3600 J/m2 in the presence of the triplet-state sensitizers acetone or 3-dimethylaminopropiophenone. Under these conditions of irradiation, which approximate solar UV, pyrimidine cyclobutane photodimers were introduced without producing any evidence of single-strand breaks or alkali-sensitive sites [i.e., no (6-4)pyrimidine-pyrimidone photoproducts]. This was confirmed by gel analysis, a T4 UV endonuclease nicking assay specific for cyclobutane-type dimers, and HPLC analysis of the photoproducts. The methylation of irradiated templates by DNA methyltransferase was inhibited in an approximately linear fashion as a function of increasing UVB dose. This inhibition was correlated with the number of lethal photoproducts detected by the simultaneous measurement of the surviving fraction of infectious phage DNA. For approximately the same number of pyrimidine cyclobutane photoproducts introduced, de novo methylation activity was approximately 2-fold more sensitive than the maintenance mode of methylation. The ability of these putatively carcinogenic, pyrimidine photoproducts to inhibit DNA methylation suggests a common mechanism of action with several chemical carcinogens that are known to modify bases.
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PMID:Perturbation of maintenance and de novo DNA methylation in vitro by UVB (280-340 nm)-induced pyrimidine photodimers. 386 17

In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transformation frequencies of oriC plasmids into E. coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation. Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase. oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells. This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase. Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E. coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity. Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin.
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PMID:Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli. 389 29

The highly purified restriction endonucleases of E. coli K and coliphage P1 transfer methyl groups from S-adenosylmethionine to adenine residues of unmodified DNA. Incubation of unmodified DNA with endonucleases K or P and S-adenosylmethionine renders the DNA resistant to restriction. The enzymes, therefore, have both restriction endonuclease and modification methylase activities.
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PMID:DNA modification methylase activity of Escherichia coli restriction endonucleases K and P. 456 4


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