Modulation of Escherichia coli DNA methyltransferase activity by biologically derived GATC-flanking sequences.
Escherichia coli DNA adenine methyltransferase (EcoDam) methylates the N-6 position of the adenine in the sequence 5'-GATC-3' and plays vital roles in gene regulation, mismatch repair, and DNA replication. It remains unclear how the small number of critical GATC sites involved in the regulation of replication and gene expression are differentially methylated, whereas the approximately 20,000 GATCs important for mismatch repair and dispersed throughout the genome are extensively methylated. Our prior work, limited to the pap regulon, showed that methylation efficiency is controlled by sequences immediately flanking the GATC sites. We extend these studies to include GATC sites involved in diverse gene regulatory and DNA replication pathways as well as sites previously shown to undergo differential in vivo methylation but whose function remains to be assigned. EcoDam shows no change in affinity with variations in flanking sequences derived from these sources, but methylation kinetics varied 12-fold. A-tracts immediately adjacent to the GATC site contribute significantly to these differences in methylation kinetics. Interestingly, only when the poly(A) is located 5' of the GATC are the changes in methylation kinetics revealed. Preferential methylation is obscured when two GATC sites are positioned on the same DNA molecule, unless both sites are surrounded by large amounts of nonspecific DNA. Thus, facilitated diffusion and sequences immediately flanking target sites contribute to higher order specificity for EcoDam; we suggest that the diverse biological roles of the enzyme are in part regulated by these two factors, which may be important for other enzymes that sequence-specifically modify DNA.
Coffin
Stephanie R
SR
Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510, USA.
Reich
Norbert O
NO
D001483:Base Sequence; D017124:Conserved Sequence; D019175:DNA Methylation; D021901:DNA, Intergenic / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D021901:DNA, Intergenic; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)