Title Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA /
Authors Lukinavičius, Gražvydas ; Lapinaitė, Audronė ; Urbanavičiūtė, Giedrė ; Gerasimaitė, Rūta ; Klimašauskas, Saulius
DOI 10.1093/nar/gks914
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Is Part of Nucleic acids research.. Oxford : Oxford University Press. 2012, Vol. 40, no 22, p. 11594-11602.. ISSN 0305-1048
Abstract [eng] 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.
Published Oxford : Oxford University Press
Type Journal article
Language English
Publication date 2012
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