In vivo analysis of the specificity of DNA adenine methyltransferase mutants R116A and N126A

Abstract

DNA adenine methyltransferase (Dam) methylates GATC sites and is important for mismatch repair and the expression of pathogenic genes in Escherichia coli (E. coli). The three base pairs flanking each GATC site influence the methylation efficiency of Dam for that site. In vitro and in vivo studies have demonstrated that methylation of GATC sites with A/T rich flanking sequences are less efficiently methylated (“bad” sites) than those with G/C rich flanking sequences (“good” sites). Our goal is to determine how the structure of Dam contributes to this preference for certain GATC sites and what residues are involved. R116 and N126 are conserved Dam residues that lie within close proximity to the GATC flanking sequences. These residues were independently mutated to alanine, forming Dam mutants R116A and N126A. We hypothesize that disruption of the hydrogen bonds between the GATC flanking sequences and the amino acid side chains of R116 or N126 will affect the methylation efficiency of Dam at “good” and “bad” GATC sites. An in vivo analysis of methylation efficiency of the mutants at various GATC sites was performed. Our preliminary results reveal that both mutants show a difference in GATC site preference compared to the wildtype, suggesting the importance of these residues in specificity. Analysis of the mutants will illuminate an alternate route for the control of bacterial DNA methylation that is crucial for pathogenesis.