The Type II restriction endonuclease MvaI has dual specificity

Stier, Ildikó; Kiss, Antal

doi:10.1093/nar/gkq676

Cited by 4 publications

(2 citation statements)

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“…Plasmids pBHNS-MSssI(F17S) and pBHNS-MSssI(G19D) encode mutant variants of M.SssI, and were created from pBHNS-MSssI by site-directed mutagenesis [23]. Plasmid pSTC-MSssI (former name pSTB-MSssI) [24] contains the gene of M.SssI (WT) in the pSC101-based plasmid vector pST76-C (Cm R ) [25] characterized by heat-sensitive replication. Plasmids pSTdC-MSssI, pSTdC-MSssI(F17S) and pSTdC-MSssI(G19D) are derivatives of pSTC-MSssI and carry the genes of untagged WT or mutant M.SssI as indicated.…”

Section: Methodsmentioning

confidence: 99%

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

Stier

Kiss

2013

PLoS ONE

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The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung + host proficient in uracil excision repair.

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Section: Methodsmentioning

confidence: 99%

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

Stier

Kiss

2013

PLoS ONE

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“…Methyl transferases methylate DNA in the genome of bacteria, to prevent its genome from being killed by its own REase. Specifically, the methylation occurs on a cytosine or an adenine in the recognition sequence to produce C5-methylcytosine, N4-methylcytosine or N6-methyladenine [2]. The protection occurs because methylated and hemimethylated sites are not recognized by the restriction endonuclease.…”

Section: Introductionmentioning

confidence: 99%

A Strategy to Purify and Express the Restriction Endonuclease BcnI

Pryce¹

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Restriction endonucleases, also known as restriction enzymes, are an essential class of enzymes that cleave the phosphodiester bond of polynucleotide chains at or near a specific nucleotide sequence. Due to the variety of mechanisms endonucleases can enact, they have been classified into four types. Type II restriction endonucleases are typically used in lab experiments because they cut DNA at defined positions producing distinct fragments and gel banding patterns. The mechanism in which these enzymes interact with DNA, specifically the differences between monomers and dimers, are unknown. Furthermore, there has not been a universal protocol made to produce different varieties of Type II restriction endonucleases so that DNA-protein interactions can be observed. We have used Infusion Cloning to insert a synthetic gene encoding the restriction endonuclease for BcnI into a plasmid and generated an expression vector for this protein of interest. Expression of the synthetic BcnI gene from the expression vector is under the control of the LacI operon and T7 promoter. Protein expression will be produced by transforming the BcnI expression vector into E. coli. This expression will be regulated through the LacI operon using glucose as well as the lactose analog IPTG to turn expression on and off. If this strategy is successful, it will yield a universal protocol for protein expression that will permit any restriction endonuclease to be studied. Here we demonstrate successful development of a BcnI expression vector construct as well as the first steps in an induction and purification protocol. This research is an essential step in a larger project that aims to quantify and describe DNA-endonuclease interactions using Förster Resonance Energy Transfer (FRET), which quantifies the energy transfer between two components, and Total Internal Reflection Fluorescence (TIRF) microscopy.

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Cloning and characterization of the TneDI restriction–modification system of Thermotoga neapolitana

Han

Cao

et al. 2011

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The Type II restriction endonuclease MvaI has dual specificity

Cited by 4 publications

References 35 publications

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

A Strategy to Purify and Express the Restriction Endonuclease BcnI

Cloning and characterization of the TneDI restriction–modification system of Thermotoga neapolitana

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