My laboratory is engaged in two areas of research:
(i) The study of the DNA-[adenine-N6] methyltransferases (MTases) encoded by bacteriophages T2 and T4, the so-called Dam MTases. Genes encoding the wild type and various mutant forms of these enzymes have been cloned, over-expressed and the corresponding proteins purified. Comparative biochemical studies with these MTases have allowed us to analyze the functional roles for various conserved motifs, the enzyme's interaction with various synthetic oligodeoxynucleotide (ODN) duplex substrates, and to investigate the reaction mechanism. In addition, X-ray crystallographic studies have recently revealed the 3-D structure of T4 Dam complexed with various substrates/products of the methylation reaction. These will aid in understanding the biochemical properties of various mutant Dam proteins.
(ii) We have begun focusing our efforts on two unicellular eukaryotic protozoa, Tetrahymena thermophila and Plasmodium falciparum (the human malaria parasite), with respect to their DNA-[N6-adenine]-MTases. Efforts are underway to clone the genes that encode them, and to determine the possible importance of DNA methylation in the growth and development of these organisms.
Graphic representation of a ternary complex between a DNA-[cytosine-5]-MTase and its substrates, DNA and methyl donor, S-adenosyl-methionine (AdoMet). The phosphodiester backbone is in magenta, the base-pairs are in orange, AdoMet is in red, and the MTase in gray. Note that the target cytosine residue is "flipped out" of the helix and into the active site of the enzyme. Base flipping is believed to be an obligatory intermediate stage in the methylation reaction characteristic of all DNA MTases.
- 2009. The first recognized epigenetic signal. DNA glucosylation of T-even bacteriophages.Epigenetics4: 151-152.
- 2009. Dimeric/oligomeric DNA methyltransferases: an unfinished story.Biol. Chem.390: 835-844.
- 2007. TStudy of bacteriophage T4-encoded Dam DNA-[adenine-N6]-methyltransferase binding by rapid laser UV cross-linking.J. Biol. Chem.282: 26067-26076.
- 2007. Differential methylation kinetics of individual target site strands by T4Dam DNA methyltransferase.Biol. Chem.388: 1199-1207.
- 2006. A probabilistic approach to compact steady-state kinetic equations for enzymatic reactions.J. Theor. Biol.242: 627-633.
- 2005. DNA-[adenine] methylation in lower eukaryotes.Biochemistry70: 550-558.
- 2005. Transition from nonspecific to specific DNA interactions along the substrate-recognition pathway of Dam methyltransferase.Cell121: 349-361.
- 2004. Bacteriophage T4Dam DNA-(adenine-N6)-methyltransferase : Comparison of pre-steady state and single turnover methylation of 40mer duplexes containing two (un)modified target sites.J. Biol. Chem.279: 50012-50018.
- 2004. Molecular enzymology of phage T4 Dam DNA methyltransferase.Mol. Biol.38: 737-751.
- 2004. Symmetry elements in DNA structure important for the recognition/methylation by DNA [amino]-methyltransferases.Nucl. Acids Res.32: 3930-3934.
- 2004. Bacteriophage T2Dam and T4Dam DNA-[N6-adenine] methyltransferases.Nucle. Acid Res. & Mol. Biol.77: 67-126.