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  • Portland Press Ltd.  (5)
  • 1
    Online Resource
    Online Resource
    Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues
    Portland Press Ltd. ; 2020
    In:  Bioscience Reports Vol. 40, No. 9 ( 2020-09-30)
    Details
    In: Bioscience Reports, Portland Press Ltd., Vol. 40, No. 9 ( 2020-09-30)
    Abstract: Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction–modification (R–M) systems and several of them are strain-specific and phase-variable. HpyAII is a novel Type IIs phase-variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni2+ over Mg2+. Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first Histidine residue of the HNH motif to Alanine does not abolish the enzymatic activity, but instead causes loss of fidelity compared with wildtype HpyAII. Previous studies have shown that mutation of the first Histidine residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. The present study provides further insights into the evolution of restriction enzymes.
    Type of Medium: Online Resource
    ISSN: 0144-8463 , 1573-4935
    URL: Article
    DOI: 10.1042/BSR20201633
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2020
    detail.hit.zdb_id: 2014993-1
    SSG: 12
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  • 2
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    Online Resource
    A nucleotide insertion between two adjacent methyltransferases in Helicobacter pylori results in a bifunctional DNA methyltransferase
    Portland Press Ltd. ; 2011
    In:  Biochemical Journal Vol. 433, No. 3 ( 2011-02-01), p. 487-495
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 433, No. 3 ( 2011-02-01), p. 487-495
    Abstract: Helicobacter pylori has a dynamic R-M (restriction–modification) system. It is capable of acquiring new R-M systems from the environment in the form of DNA released from other bacteria or other H. pylori strains. Random mutations in R-M genes can result in non-functional R-M systems or R-M systems with new properties. hpyAVIAM and hpyAVIBM are two solitary DNA MTase (methyltransferase) genes adjacent to each other and lacking a cognate restriction enzyme gene in H. pylori strain 26695. Interestingly, in an Indian strain D27, hpyAVIAM–hpyAVIBM encodes a single bifunctional polypeptide due to insertion of a nucleotide just before the stop codon of hpyAVIBM and, when a similar mutation was made in hpyAVIAM–hpyAVIBM from strain 26695, a functional MTase with an N-terminal C5-cytosine MTase domain and a C-terminal N6-adenine MTase domain was constructed. Mutations in the AdoMet (S-adenosylmethionine)-binding motif or in the catalytic motif of M.HpyAVIA or M.HpyAVIB selectively abrogated the C5-cytosine or N6-adenine methylation activity of M.HpyAVIA–M.HpyAVIB fusion protein. The present study highlights the ability of H. pylori to evolve genes with unique functions and thus generate variability. For organisms such as H. pylori, which have a small genome, these adaptations could be important for their survival in the hostile host environment.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20101668
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2011
    detail.hit.zdb_id: 1473095-9
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    Structure, function and mechanism of exocyclic DNA methyltransferases
    Portland Press Ltd. ; 2006
    In:  Biochemical Journal Vol. 399, No. 2 ( 2006-10-15), p. 177-190
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 399, No. 2 ( 2006-10-15), p. 177-190
    Abstract: DNA MTases (methyltransferases) catalyse the transfer of methyl groups to DNA from AdoMet (S-adenosyl-L-methionine) producing AdoHcy (S-adenosyl-L-homocysteine) and methylated DNA. The C5 and N4 positions of cytosine and N6 position of adenine are the target sites for methylation. All three methylation patterns are found in prokaryotes, whereas cytosine at the C5 position is the only methylation reaction that is known to occur in eukaryotes. In general, MTases are two-domain proteins comprising one large and one small domain with the DNA-binding cleft located at the domain interface. The striking feature of all the structurally characterized DNA MTases is that they share a common core structure referred to as an ‘AdoMet-dependent MTase fold’. DNA methylation has been reported to be essential for bacterial virulence, and it has been suggested that DNA adenine MTases (Dams) could be potential targets for both vaccines and antimicrobials. Drugs that block Dam could slow down bacterial growth and therefore drug-design initiatives could result in a whole new generation of antibiotics. The transfer of larger chemical entities in a MTase-catalysed reaction has been reported and this represents an interesting challenge for bio-organic chemists. In general, amino MTases could therefore be used as delivery systems for fluorescent or other reporter groups on to DNA. This is one of the potential applications of DNA MTases towards developing non-radioactive DNA probes and these could have interesting applications in molecular biology. Being nucleotide-sequence-specific, DNA MTases provide excellent model systems for studies on protein–DNA interactions. The focus of this review is on the chemistry, enzymology and structural aspects of exocyclic amino MTases.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20060854
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2006
    detail.hit.zdb_id: 1473095-9
    SSG: 12
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  • 4
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    Online Resource
    The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL
    Portland Press Ltd. ; 2009
    In:  Biochemical Journal Vol. 423, No. 2 ( 2009-10-15), p. 265-277
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 423, No. 2 ( 2009-10-15), p. 265-277
    Abstract: The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn2+, Mg2+ or Ca2+ ions, unlike human MutLα which shows endonuclease activity only in the presence of Mn2+. We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460–658 exhibits Mn2+-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX2EX4E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX2EX4E motif.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20090626
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2009
    detail.hit.zdb_id: 1473095-9
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis
    Portland Press Ltd. ; 2008
    In:  Biochemical Journal Vol. 410, No. 3 ( 2008-03-15), p. 543-553
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 410, No. 3 ( 2008-03-15), p. 543-553
    Abstract: Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5′-CAGCAG-3′. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20070900
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2008
    detail.hit.zdb_id: 1473095-9
    SSG: 12
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