| DNA methylation is a ubiquitous process where methyl groups from the donor S-adenosylmethionine (AdoMet) are transferred to either adenine or cytosine DNA bases in both prokaryotes and eukaryotes. Failures of the normal function of this system are linked to genetic disorders, cancer, lupus, and loss of bacterial virulence. The most thoroughly studied DNA methylation systems are from bacterial restriction-modification systems. I have been investigating structure-function relationships in the Rsrl modification DNA methyltransferase (M.RsrI) from Rhodobacter sphaeroides, which acts on the double-stranded DNA sequence GAATTC.; I determined the X-ray crystal structure of the M.RsrI, which revealed the enzyme bound an unexpected ligand, 5-methylthioadenosine, in the ligand-binding site. In addition, the protein appeared to be a homodimer. Subsequently, I determined the crystal structures of the methyltransferase with bound S-adenosylmethionine, S-adenosylhomocysteme (reaction product), or sinefungin (inhibitor) in the ligand binding site. Comparisons of these structures identified a unique binding orientation for AdoMet. I also determined the structure of the L72P mutant of M.RsrI, isolated previously as a catalytically-impaired, DNA-binding mutant, and observed the first methyltransferase structure lacking both bound ligand and DNA.; Since most methyltransferases studied function as monomers, we investigated whether the dimerization observed in the crystal structure was biochemically important. Gel-filtration chromatography of M.RsrI revealed a single peak with the molecular weight of a dimer and chromatography of M.RsrI bound to DNA suggested the complex consisted of a dimeric enzyme bound to a single DNA molecule.; Comparison of the dimer interface of M.RsrI with the recent M.MboII structure and sequence alignments allowed identification of a conserved dimerization motif. I disrupted the dimerization of M.RsrI by adding N-acetyl-phenylalanine as a competitive inhibitor and by mutation of a serine located in the dimer interface to an aspartate. Both methods resulted in inhibition of the enzyme activity, suggesting dimerization is important for enzyme activity. |
