Cloning and characterization of the<i>Hpall</i>methylase gene

Card, Charles O.; Wilson, Geoffrey G.; Weule, Karin; Hasapes, Joseph P.; Kiss, Antal; Roberts, Richard J.

doi:10.1093/nar/18.6.1377

Cited by 38 publications

(17 citation statements)

References 50 publications

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“…We first turned to the HpaII C5-MTase (M.HpaII), which recognizes C C GG targets in DNA (27) and which thus can potentially be useful to study mammalian DNA modification at CpG sites. As expected, the WT enzyme showed low or no activity with AdoMet analogs 2 and 4 , respectively.…”

Section: Resultsmentioning

confidence: 99%

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

Lukinavičius

Lapinaitė

Urbanavičiūtė

et al. 2012

Nucleic Acids Research

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DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA–M.HhaI–AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA.

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Section: Resultsmentioning

confidence: 99%

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

Lukinavičius

Lapinaitė

Urbanavičiūtė

et al. 2012

Nucleic Acids Research

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“…SmaI endonuclease protection assay. Expression and activity of the recombinant M.SpyI was tested using a combination of protocols described previously (11,29) following the instructions for the pBADTOPO TA Expression Kit. Briefly, recombinant M.SpyI expression was induced by the addition of 0.2% or 2% L-arabinose to cultures at an optical density at 600 nm of 0.5, and growth then continued for 2 or 4 h at 37°C, respectively.…”

Section: Methodsmentioning

confidence: 99%

M.SpyI, a DNA Methyltransferase Encoded on a mefA Chimeric Element, Modifies the Genome of Streptococcus pyogenes

et al. 2007

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“…130 Whether this exclusion occurs through chromosomal cleavage and is accompanied by cell death, as for McrBC, is not known. An mcrA homologue of Burkholderia sp.…”

Section: Other Cases Suggesting Conflicts Involving Epigenetic Systemsmentioning

confidence: 99%

Conflicts Targeting Epigenetic Systems and Their Resolution by Cell Death: Novel Concepts for Methyl-Specific and Other Restriction Systems

2010

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Epigenetic modification of genomic DNA by methylation is important for defining the epigenome and the transcriptome in eukaryotes as well as in prokaryotes. In prokaryotes, the DNA methyltransferase genes often vary, are mobile, and are paired with the gene for a restriction enzyme. Decrease in a certain epigenetic methylation may lead to chromosome cleavage by the partner restriction enzyme, leading to eventual cell death. Thus, the pairing of a DNA methyltransferase and a restriction enzyme forces an epigenetic state to be maintained within the genome. Although restriction enzymes were originally discovered for their ability to attack invading DNAs, it may be understood because such DNAs show deviation from this epigenetic status. DNAs with epigenetic methylation, by a methyltransferase linked or unlinked with a restriction enzyme, can also be the target of DNases, such as McrBC of Escherichia coli, which was discovered because of its methyl-specific restriction. McrBC responds to specific genome methylation systems by killing the host bacterial cell through chromosome cleavage. Evolutionary and genomic analysis of McrBC homologues revealed their mobility and wide distribution in prokaryotes similar to restriction–modification systems. These findings support the hypothesis that this family of methyl-specific DNases evolved as mobile elements competing with specific genome methylation systems through host killing. These restriction systems clearly demonstrate the presence of conflicts between epigenetic systems.

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Cloning and characterization of theHpallmethylase gene

Cited by 38 publications

References 50 publications

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

M.SpyI, a DNA Methyltransferase Encoded on a mefA Chimeric Element, Modifies the Genome of Streptococcus pyogenes

Conflicts Targeting Epigenetic Systems and Their Resolution by Cell Death: Novel Concepts for Methyl-Specific and Other Restriction Systems

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