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 restriction endonuclease from E. coli B is both an endonuclease and a DNA methylase. Both activities either require or are stimulated by Mg(+2), adenosine triphosphate, and S-adenosyl-L-methionine. The particular activity which the enzyme exhibits depends upon the nature of the SB sites, the genetic sites that identify substrate DNA. Enzymatic treatment of DNA that has an unmodified, wild-type SB site results in either rapid restriction of the DNA or very slow methylation of the SB site. On the other hand, a hybrid SB site (modified), which protects the DNA molecule from restriction, results in rapid methylation of that SB site.
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PMID:Kinetics of methylation of DNA by a restriction endonuclease from Escherichia coli B. 461 May 61

The gene coding for the pneumococcal DNA adenine methylase that recognizes the sequence 5'-GATC-3' was cloned in a strain of Streptococcus pneumoniae that lacked both restriction endonucleases DpnI and DpnII. The gene was cloned as a 3.7-kilobase fragment of chromosomal DNA from a DpnII-containing strain inserted in both possible orientations in the multicopy plasmid vector pMP5 to give recombinant plasmids pMP8 and pMP10. Recombinant plasmids were selected by their resistance to DpnII cleavage. Cells carrying the recombinant plasmids modified phage in vivo so that it was restricted by DpnI- but not DpnII-containing hosts. They also showed levels of DNA methylase activity five times higher than that in cells of the original DpnII strain. No DpnII activity was observed in the clones; therefore, it was concluded that the insert did not contain an intact DpnII endonuclease gene and that methylation of host DNA did not turn on a latent form of the gene.
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PMID:Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase. 632 45

Plasmid transfer via the transformation pathway of Streptococcus pneumoniae was weakly restricted by the DpnI or DpnII restriction endonuclease, either of which gave a reduction only to 0.4, compared with phage infection, which was restricted to 10(-5). The greater sensitivity of plasmid transfer compared with chromosomal transformation, which was not at all restricted, can be attributed to partially double-stranded intermediates formed from two complementary donor fragments. However, clustering of potential restriction sites in the plasmids increased the probability of escape from restriction. The recombinant plasmid pMP10 , in which the gene for the DpnII DNA methylase was cloned, can be transferred to strains that contain neither restriction enzyme or that contain DpnII as readily as can the vector pMP5 . Introduction of pMP10 raised the level of methylase by five times the level normally present in DpnII strains. Transfer of pMP10 to DpnI -containing strains was infrequent, presumably owing to the suicidal methylation of DNA which rendered it susceptible to the host endonuclease. The few clones in which pMP10 was established had lost DpnI . Loss of the plasmid after curing of the cell eliminated the methylase but did not restore DpnI . Although this loss of DpnI could result from spontaneous mutation, its relatively high frequency, 0.1% suggested that the loss was due to a regulatory shift.
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PMID:Transfer of recombinant plasmids containing the gene for DpnII DNA methylase into strains of Streptococcus pneumoniae that produce DpnI or DpnII restriction endonucleases. 632 47

A DNA methylase was purified in a homogeneous state from a extremely thermophilic bacterium, Thermus thermophilus HB8, by chromatography on, successively, phosphocellulose, CM-cellulose, and heparin-Sepharose. The molecular weight of the enzyme was determined to be about 44,000 by gel filtration on a Sephadex G-100 column and 41,000 by SDS-poly-acrylamide gel electrophoresis, and these findings suggest a single polypeptide enzyme. The enzyme develops maximum activity around pH 7.4 and at 70 degrees C. Enzymatic activity is completely inhibited by 0.2 M NaCl or 2 mM HgCl2. The enzyme transfers methyl groups from S-adenosyl-L-methionine to a double stranded DNA. The sole product of the reaction was identified as N-6-methyl adenine after hydrolysis of the DNA with formic acid. The enzyme kinetics obey the Michaelis-Menten equation and Km values for S-adenosylmethionine and lambda phage DNA were determined to be 0.8 muM and 10 microgram/ml, respectively. The enzyme does not transfer methyl groups to TthHB8I endonuclease digested DNA as well as the host (T. thermophilus HB8) DNA. The number of methyl groups of the fully methylated phiX174 RF DNA was about twice as many as TthHB8I endonuclease sites on the DNA. The distribution of the methyl groups of phiX174 RF DNA among the HaeIII fragments was the same as that of TthHB8I endonuclease sites, suggesting that this DNA methylase is the other component of the modification-restriction system including TthHB8I endonuclease. The enzyme probably recognizes the sequence, 5'-TCGA-3', in a double stranded DNA and probably methylates adenine in the above sequence.
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PMID:A DNA methylase from Thermus thermophilus HB8. 644 53

Oxidation of DNA and its components can cause genetic mutations and chromosomal instability. These changes have generally been implicated in aging. Oxidation of the methyl group of thymidine residues in DNA is known to result in the formation 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd). We have utilized Bacillus subtilis phage SPO1 DNA as a model of oxidatively damaged DNA. In this phage, all thymine (Thy) residues are replaced by 5-hydroxymethyluracil (5HmUra), but the species is naturally devoid of other oxidatively-induced DNA lesions. Particular attention was paid to the behavior of 5HmUra-containing DNA as a target for several enzymes employing DNA as substrate; restriction endonucleases, dam DNA methylase and T4 DNA ligase. We noticed that susceptibility of SPO1 DNA varied when different restriction endonucleases having 5HmUra in the restriction sites were tested. Endonucleolytic cleavage brought about Sau3A proceeded as effectively with SPO1 DNA as with conventional DNA (lambda phage). The same was true when the ligation of Sau3A sites was performed with T4 DNA ligase. In contrast, both endonucleolytic cleavage and ligation were slower in SPO1 DNA, compared with lambda phage, when Taq I and T4 DNA ligase were used for restriction and ligation, respectively. We also noticed that SPO1 phage does not naturally contain N6-methyladenine (N6MeAde) opposite 5HmUra, i.e., no hydrolysis of SPO1 DNA was observed when assessed with methylation-dependent restriction endonuclease DpnI. Our results show that the presence of 5HmUra in the respective site of DNA does not, per se, prevent the activity of restriction endonucleases, ligases or DNA methylases. These data support the view that oxidation of Thy to 5HmUra in target DNA does not necessarily result in substantial deterioration in the functions of DNA processing enzymes.
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PMID:Restriction, methylation and ligation of 5-hydroxymethyluracil-containing DNA. 786 75

Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.
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PMID:Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox. 920 41

Some genetic markers of E. coli HB101 and JM110 were identified, two bacterial strains were used as recipients respectively to detect the expression of a restriction endonuclease(R) gene and a methylase(M) gene of BstNI isoschizomer restriction-modification system. DNA fragment containing the R-M genes was deleted unilaterally with exoIII and 23 deletion subclones were obtained. According to the Enzyme activity of each subclone, R and M gene were located respectively at the regions of 0.2-->1.4 kb and 1.5-->3.3 kb from cloning site PstI. Analysis showed that the R. M system belongs to type II, two genes are controlled by the different promoters; the recognition sequence of this system is the same as that of DNA-cytosine methyltransferase(Dcm), the latter's methylation function can resist the R enzyme. It was interesting that the recombinant plasmid with an R+ M- genotype appeared to be lethal to dcm+ hosts yet. This indicated that the M gene closely linking to R gene is of critical importance for the existence of the R-M system in process of evolution.
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PMID:[Expression detection and location analysis of BstNI isoschizomer restriction-modification system gene]. 1255 36

DNA methylation at the 5th position of cytosine has been found to be correlated with tumorigenesis. An inhibitor of DNA methylase could, therefore, be used as an anticancer drug. However, only a few inhibitory compounds have been discovered due to the limitations for assaying the DNA methylation. In this study, we describe a modification of DNA cytosine-C5-methyltransferase assay system utilizing [(3)H]-labeled S-adenosyl-methionine (SAM) and Sephadex G-25 column. Pre-treatment of either lambda DNA or the promoter region of human telomerase (hTERT) with HaeIII methylase greatly reduced the digestion of the DNAs with the corresponding restriction enzyme HaeIII endonuclease (over 100-fold), and the result was further confirmed by agarose gel electrophoresis. Application of this column method to another modification/restriction system, EcoRI methylase/endonuclease, gave rise to the similar results. Our data suggest that the newly developed column method could be effective for rapid screening of large number of cytosine methylase inhibitors and could also be applicable to other DNA methylases.
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PMID:A column method for determination of DNA cytosine-C5-methyltransferase activity. 1476 31

Virion DNA of bacteriophage 11b (Phi11b), which infects a psychrophilic Flavobacterium isolate from Arctic sea-ice, was determined to consist of 36,012 bp. With 30.6% its GC content corresponds to that of host-genus species and is the lowest of all phages of Gram-negative bacteria sequenced so far. Similarities of several of 65 predicted ORFs, genome organization and phylogeny suggest an affiliation to 'mesophilic' nonmarine siphoviruses, e.g. to bacteriophages SPP1 and HK97. Early genes presumably encode an essential recombination factor (ERF), a single strand binding (SSB) protein, an endonuclease, and a DNA methylase. The late gene segment is likely to contain a terminase, portal, minor head, protease and a major capsid gene. Five ORFs exhibited similarities to Bacteroidetes species and seem to reflect the host specificity of the phage. Among PAGE-separated virion proteins that were identified by MALDI-ToF mass spectrometry are the portal, the major capsid, and a putative conserved tail protein. The Phi11b genome is the first to be described of a cultivated virus infecting a psychrophilic host as well as a Bacteroidetes bacterium.
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PMID:Genome and proteome characterization of the psychrophilic Flavobacterium bacteriophage 11b. 1693 43

Some adenine methyltransferases have been shown not only to protect specific DNA restriction sites from cleavage by a restriction endonuclease, but also to play a role in various bacterial processes and sometimes in bacterial virulence. This study focused on a type I restriction-modification system (designated yrmI) of Y. pseudotuberculosis. This system is composed of three adjacent genes which could potentially encode an N6-adenine DNA methylase (YamA), an enzyme involved in site-specific recognition (YrsA) and a restriction endonuclease (YreA). Screening of 85 isolates of Y. pestis and Y. pseudotuberculosis indicated that the yrmI system has been lost by Y. pestis and that yamA (but not yrsA or yreA) is present in all Y. pseudotuberculosis strains tested, suggesting that it may be important at some stages of the epidemiological cycle of this species. To further investigate the role of yamA in Y. pseudotuberculosis survival, multiplication or virulence, a DeltayamA mutant of Y. pseudotuberculosis IP32953 was constructed by allelic exchange with a kanamycin cassette. The fact that DeltayamA mutants were obtained indicated that this gene is not essential for Y. pseudotuberculosis viability. The IP32953DeltayamA mutant strain grew as well as the wild-type in a rich medium at both 28 degrees C and 37 degrees C. It also grew normally in a chemically defined medium at 28 degrees C, but exhibited a growth defect at 37 degrees C. In contrast to the Dam adenine methyltransferase, a mutation in yamA did not impair the functions of DNA repair or resistance to detergents. However, the DeltayamA mutant exhibited a virulence defect in a mouse model of intragastric infection. The in silico analysis indicated that the chromosomal region carrying the Y. pseudotuberculosis yrmI locus has been replaced in Y. pestis by a horizontally acquired region which potentially encodes another methyltransferase. YamA might thus be dispensable for Y. pestis growth and virulence because this species has acquired another gene fulfilling the same functions.
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PMID:A putative DNA adenine methyltransferase is involved in Yersinia pseudotuberculosis pathogenicity. 1766 Apr 7


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