Modulation of Escherichia coli DNA Methyltransferase Activity by Biologically Derived GATC-flanking Sequences

Abstract

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 ϳ20,000 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.

Escherichia coli DNA Adenine Methyltransferase

“…Furthermore, the orthologous residues in T4 Dam have been shown to stabilize a nonspecific enzyme-DNA complex (29). This observation, in addition to our previous work, suggests that these residues may aid in the processive methylation of multiple GATC sites in EcoDam in addition to providing an indirect readout mechanism responsible for higher-order specificity (14,25).…”

“…However, the R95A, R116A, N132A, and N126A all show an up to 10-fold decrease in processivity and 25-fold enhancement of the preference for the preferred GATC over the non-preferred GATC. completion) we can describe the fraction of enzymatic encounters that result in double methylation events as the processivity factor (f P ) and the site preference on this substrate (E P /E N-P ) as previously described (25)(26)(27). For example, Stanford et al (27) demonstrated that the endonuclease EcoRV endonuclease is less likely to processively cleave adjacent cognate sites as the distance between them increases.…”

“…To identify the kinetic step responsible for the poor processivity of R116A, N126A, R95A, and N132A, we kinetically characterized all mutants using two DNA substrates based on the preferential or non-preferential methylation by EcoDam (25). An A-tract positioned 5Ј to the GATC (N-P) was shown to decrease methylation kinetics and exacerbate preferential methylation for WT EcoDam when compared with a GATC without an A-tract (P).…”

“…We previously showed that EcoDam processivity and differential methylation of biologically derived GATC sites is in part regulated by the DNA immediately flanking the target site (14,25). This "higher order" specificity modulates the ability of the enzyme to methylate a particular sequence and act processively, and is important to the function of the enzyme in several biological pathways.…”

“…3 This work offers insight into the structural basis of processive catalysis for an enzyme that does not completely enclose the DNA in addition to enhancing our understanding of indirect read-out by DNA modifying enzymes. (25). In brief, cells containing the desired construct were grown at 37°C in LB media supplemented with 25 g/ml kanamycin and 12.5 g/ml tetracycline.…”