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

Lukinavicius G, Lapinaite A, Urbanaviciute G, Gerasimaite R, Klimasauskas S - Nucleic Acids Res. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, 02241 Vilnius, Lithuania.

ABSTRACT
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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Homology-based engineering of C5-MTases in conserved sequence motifs IV and X. (a) Amino acid sequence alignment of regions corresponding to IV and X conserved motifs of prokaryotic and eukaryotic cytosine-C5 MTases. Arrows indicate Gln82 and Asn304 positions of M.HhaI in IV and X conserved motifs. Four degrees of conservation in descending order: black background with white text, dark gray background with white text, gray background with black text and white background with black text. Amino acid conservation of the Gln82 position (HhaI) in DNA C5-MTases is depicted, based on an alignment of ∼530 DNA C5-MTase sequences from the Pfam database (http:www.sanger.ac.uk/Software/Pfam/) (b) Permutation of conserved motifs in the M.HhaI, M.HpaII and M2.Eco31I DNA methyltransferases. (c) Enzymatic transalkylation of DNA by engineered variants of M.HpaII and M2.Eco31I. Turnover rates (shown in logarithmic scale) were estimated using the DNA protection assay.
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gks914-F6: Homology-based engineering of C5-MTases in conserved sequence motifs IV and X. (a) Amino acid sequence alignment of regions corresponding to IV and X conserved motifs of prokaryotic and eukaryotic cytosine-C5 MTases. Arrows indicate Gln82 and Asn304 positions of M.HhaI in IV and X conserved motifs. Four degrees of conservation in descending order: black background with white text, dark gray background with white text, gray background with black text and white background with black text. Amino acid conservation of the Gln82 position (HhaI) in DNA C5-MTases is depicted, based on an alignment of ∼530 DNA C5-MTase sequences from the Pfam database (http:www.sanger.ac.uk/Software/Pfam/) (b) Permutation of conserved motifs in the M.HhaI, M.HpaII and M2.Eco31I DNA methyltransferases. (c) Enzymatic transalkylation of DNA by engineered variants of M.HpaII and M2.Eco31I. Turnover rates (shown in logarithmic scale) were estimated using the DNA protection assay.

Mentions: The structural conservation of C5-MTases and successful engineering of M.HhaI suggested that other orthologs can be similarly engineered based on sequence alignment even in the absence of crystal structures (Figure 6). We first turned to the HpaII C5-MTase (M.HpaII), which recognizes CCGG 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. Replacement of the Q104 and N335 residues (correspond to Q82 and N304 in M.HhaI) to alanines resulted in an enzyme with a significantly enhanced activity toward these analogs (Figure 6, Supplementary Figure S1B and Supplementary Table S3).Figure 6.


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

Lukinavicius G, Lapinaite A, Urbanaviciute G, Gerasimaite R, Klimasauskas S - Nucleic Acids Res. (2012)

Homology-based engineering of C5-MTases in conserved sequence motifs IV and X. (a) Amino acid sequence alignment of regions corresponding to IV and X conserved motifs of prokaryotic and eukaryotic cytosine-C5 MTases. Arrows indicate Gln82 and Asn304 positions of M.HhaI in IV and X conserved motifs. Four degrees of conservation in descending order: black background with white text, dark gray background with white text, gray background with black text and white background with black text. Amino acid conservation of the Gln82 position (HhaI) in DNA C5-MTases is depicted, based on an alignment of ∼530 DNA C5-MTase sequences from the Pfam database (http:www.sanger.ac.uk/Software/Pfam/) (b) Permutation of conserved motifs in the M.HhaI, M.HpaII and M2.Eco31I DNA methyltransferases. (c) Enzymatic transalkylation of DNA by engineered variants of M.HpaII and M2.Eco31I. Turnover rates (shown in logarithmic scale) were estimated using the DNA protection assay.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526304&req=5

gks914-F6: Homology-based engineering of C5-MTases in conserved sequence motifs IV and X. (a) Amino acid sequence alignment of regions corresponding to IV and X conserved motifs of prokaryotic and eukaryotic cytosine-C5 MTases. Arrows indicate Gln82 and Asn304 positions of M.HhaI in IV and X conserved motifs. Four degrees of conservation in descending order: black background with white text, dark gray background with white text, gray background with black text and white background with black text. Amino acid conservation of the Gln82 position (HhaI) in DNA C5-MTases is depicted, based on an alignment of ∼530 DNA C5-MTase sequences from the Pfam database (http:www.sanger.ac.uk/Software/Pfam/) (b) Permutation of conserved motifs in the M.HhaI, M.HpaII and M2.Eco31I DNA methyltransferases. (c) Enzymatic transalkylation of DNA by engineered variants of M.HpaII and M2.Eco31I. Turnover rates (shown in logarithmic scale) were estimated using the DNA protection assay.
Mentions: The structural conservation of C5-MTases and successful engineering of M.HhaI suggested that other orthologs can be similarly engineered based on sequence alignment even in the absence of crystal structures (Figure 6). We first turned to the HpaII C5-MTase (M.HpaII), which recognizes CCGG 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. Replacement of the Q104 and N335 residues (correspond to Q82 and N304 in M.HhaI) to alanines resulted in an enzyme with a significantly enhanced activity toward these analogs (Figure 6, Supplementary Figure S1B and Supplementary Table S3).Figure 6.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, 02241 Vilnius, Lithuania.

ABSTRACT
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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