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

Nucleic Acids Res. 2012 Dec;40(22):11594-602. doi: 10.1093/nar/gks914. Epub 2012 Oct 5.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkyl and Aryl Transferases / chemistry
  • Alkyl and Aryl Transferases / genetics*
  • Alkyl and Aryl Transferases / metabolism
  • Alkylation
  • Amino Acid Sequence
  • Catalytic Domain
  • DNA / chemistry
  • DNA / metabolism*
  • DNA-Cytosine Methylases / chemistry
  • DNA-Cytosine Methylases / genetics*
  • DNA-Cytosine Methylases / metabolism*
  • Escherichia coli / genetics
  • Molecular Sequence Data
  • Mutation
  • Protein Engineering
  • S-Adenosylmethionine / metabolism
  • Sequence Alignment
  • Site-Specific DNA-Methyltransferase (Adenine-Specific) / chemistry
  • Site-Specific DNA-Methyltransferase (Adenine-Specific) / genetics
  • Site-Specific DNA-Methyltransferase (Adenine-Specific) / metabolism

Substances

  • S-Adenosylmethionine
  • DNA
  • DNA modification methylase Eco31I
  • DNA modification methylase HhaI
  • DNA-Cytosine Methylases
  • Site-Specific DNA-Methyltransferase (Adenine-Specific)
  • Alkyl and Aryl Transferases
  • DNA alkyltransferase