Limits...
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.

Show MeSH

Related in: MedlinePlus

mTAG aminoalkylation and labeling of DNA in the presence of AdoMet. pUC19 plasmid was modified with M.HhaI (Q82A/Y254S/N304A mutant) and a mixture of AdoMet and cofactor 4 supplied in different molar fractions as indicated (total cofactor concentration was 50 µM). Modified DNA was labeled with Cy5-NHS, fragmented with R.BseSI and separated by agarose gel electrophoresis. Bulk DNA was visualized after staining with EtBr. Visualization was performed using 473 nm (EtBr) and 635 nm (Cy5) lasers. C is control sample incubated without cofactor; M, DNA size marker GeneRuler™ 100 bp Plus DNA Ladder.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks914-F4: mTAG aminoalkylation and labeling of DNA in the presence of AdoMet. pUC19 plasmid was modified with M.HhaI (Q82A/Y254S/N304A mutant) and a mixture of AdoMet and cofactor 4 supplied in different molar fractions as indicated (total cofactor concentration was 50 µM). Modified DNA was labeled with Cy5-NHS, fragmented with R.BseSI and separated by agarose gel electrophoresis. Bulk DNA was visualized after staining with EtBr. Visualization was performed using 473 nm (EtBr) and 635 nm (Cy5) lasers. C is control sample incubated without cofactor; M, DNA size marker GeneRuler™ 100 bp Plus DNA Ladder.

Mentions: Taking together the enhanced catalytic power of the engineered M.HhaI variants in alkyltransfer reactions (especially with cofactors 2 and 3) and their reduced affinity toward the natural cofactor one might expect a shifted or reversed cofactor preference during reaction. Efficient aminoalkylation reactions in the presence of endogenous AdoMet would potentially permit in situ or even in vivo derivatization and subsequent labeling of DNA. Our DNA protection experiments indicated that the turnover rates of the triple (Q82A/Y254S/N304A) mutant were similar with AdoMet or the Ado-11-amine cofactor 4 (Figure 2C), however the cofactor preference of the enzyme in direct competition between the two cofactors remained unclear. As a proof of principle, enzymatic modification of pUC19 plasmid DNA in the presence of varied molar ratios of AdoMet to its analog was performed. Methylation and alkylation of DNA was probed using combination of the R.Hin6I and R.BspLI endonucleases (Supplementary Figure S5). Moreover, incorporation of the extended chain with a reactive amino group was assessed by labeling the modified DNA with Cy5-NHS (Figure 4). Visualization of Cy5-labeled DNA showed that Q82A/Y254S/N304A variant is capable of efficiently alkylating DNA in presence of physiologically relevant concentrations of AdoMet (8.5–300 µM) (24). The label is incorporated even in presence of 5-fold excess of AdoMet (Figure 4). This result opens the possibility for sequence-specific labeling of DNA in vivo or in the cell in presence of endogenous AdoMet.Figure 4.


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)

mTAG aminoalkylation and labeling of DNA in the presence of AdoMet. pUC19 plasmid was modified with M.HhaI (Q82A/Y254S/N304A mutant) and a mixture of AdoMet and cofactor 4 supplied in different molar fractions as indicated (total cofactor concentration was 50 µM). Modified DNA was labeled with Cy5-NHS, fragmented with R.BseSI and separated by agarose gel electrophoresis. Bulk DNA was visualized after staining with EtBr. Visualization was performed using 473 nm (EtBr) and 635 nm (Cy5) lasers. C is control sample incubated without cofactor; M, DNA size marker GeneRuler™ 100 bp Plus DNA Ladder.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks914-F4: mTAG aminoalkylation and labeling of DNA in the presence of AdoMet. pUC19 plasmid was modified with M.HhaI (Q82A/Y254S/N304A mutant) and a mixture of AdoMet and cofactor 4 supplied in different molar fractions as indicated (total cofactor concentration was 50 µM). Modified DNA was labeled with Cy5-NHS, fragmented with R.BseSI and separated by agarose gel electrophoresis. Bulk DNA was visualized after staining with EtBr. Visualization was performed using 473 nm (EtBr) and 635 nm (Cy5) lasers. C is control sample incubated without cofactor; M, DNA size marker GeneRuler™ 100 bp Plus DNA Ladder.
Mentions: Taking together the enhanced catalytic power of the engineered M.HhaI variants in alkyltransfer reactions (especially with cofactors 2 and 3) and their reduced affinity toward the natural cofactor one might expect a shifted or reversed cofactor preference during reaction. Efficient aminoalkylation reactions in the presence of endogenous AdoMet would potentially permit in situ or even in vivo derivatization and subsequent labeling of DNA. Our DNA protection experiments indicated that the turnover rates of the triple (Q82A/Y254S/N304A) mutant were similar with AdoMet or the Ado-11-amine cofactor 4 (Figure 2C), however the cofactor preference of the enzyme in direct competition between the two cofactors remained unclear. As a proof of principle, enzymatic modification of pUC19 plasmid DNA in the presence of varied molar ratios of AdoMet to its analog was performed. Methylation and alkylation of DNA was probed using combination of the R.Hin6I and R.BspLI endonucleases (Supplementary Figure S5). Moreover, incorporation of the extended chain with a reactive amino group was assessed by labeling the modified DNA with Cy5-NHS (Figure 4). Visualization of Cy5-labeled DNA showed that Q82A/Y254S/N304A variant is capable of efficiently alkylating DNA in presence of physiologically relevant concentrations of AdoMet (8.5–300 µM) (24). The label is incorporated even in presence of 5-fold excess of AdoMet (Figure 4). This result opens the possibility for sequence-specific labeling of DNA in vivo or in the cell in presence of endogenous AdoMet.Figure 4.

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.

Show MeSH
Related in: MedlinePlus