Conformational Transitions as Determinants of Specificity for the DNA Methyltransferase EcoRI

Abstract

19269 -19275). The direct measurement of the kinetic rate constants for DNA bending, intercalation, and base flipping with cognate and noncognate substrates (GAATTT, GGATTC) of wild type M.EcoRI using fluorescence resonance energy transfer and 2-aminopurine fluorescence studies reveals that DNA bending precedes both intercalation and base flipping, and base flipping precedes intercalation. Destabilization of these intermediates provides a molecular basis for understanding how conformational transitions contribute to specificity. The 3500-and 23,000-fold decreases in sequence specificity for noncognate sites GAATTT and GGATTC are accounted for largely by an ϳ2500-fold increase in the reverse rate constants for intercalation and base flipping, respectively. Thus, a predominant contribution to specificity is a partitioning of enzyme intermediates away from the Michaelis complex prior to catalysis. Our results provide a basis for understanding enzyme specificity and, in particular, sequence-specific DNA modification. Because many DNA methyltransferases and DNA repair enzymes induce similar DNA distortions, these results are likely to be broadly relevant.DNA-modifying enzymes have evolved a delicate balance between sequence specificity and efficient catalysis (1, 2). Indeed, the lack of our understanding of the underlying causes for specificity may account in part for the general lack of success in re-engineering such enzymes, despite relative successes with other classes of enzymes (3-5). Many DNA-modifying enzymes have specificity constants that approach the predicted values for diffusion-controlled reactions (k cat /K m ϳ 10 8 -10 9 M Ϫ1 s Ϫ1 ) (6, 7). DNA-modifying enzymes have attained such efficiency in part because of facilitated diffusion mechanisms (8). Remarkably, such highly efficient enzymes also show significant discrimination between cognate and noncognate sequences.These enzymes frequently undergo significant conformational changes upon binding their cognate sequence, a clear example of an induced fit mechanism. The resultant intermediates provide a theoretical basis for modulating specificity through the concept of kinetic proofreading, as proposed for several DNA polymerases (9). These checkpoints can directly impact specificity by modifying the partitioning of reaction intermediates prior to catalysis differentially between cognate and noncognate substrates (9 -11).S-Adenosylmethionine-dependent bacterial DNA methyltransferases modify bases at cytosine (C-5 and N-4) and adenine (N-6) and play important roles in many cellular pathways, including mismatch repair, restriction modification, and gene regulation (12). A detailed structural and mechanistic characterization of DNA methyltransferases is also motivated by recent demonstrations that some bacterial and mammalian enzymes are viable candidates for antibiotic and cancer drug development, respectively (13-16). The bacterial DNA adenine methyltransferase EcoRI forms part of a restriction/modification system and modifies the N-6 position of the seco...

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

“…However, the original observations by Guschlbauer and co-workers (28) suggest a more dramatic variation in preferential methylation on GATC sites within large plasmid substrates. We have observed differential kinetics between small and large substrates with other DNA-modifying enzymes (34,(61)(62)(63) and therefore sought to determine if a more biologically relevant substrate involving multiple sites and larger DNA would enhance the site preference we observed with kinetics. Our initial competition experiment placed preferred and nonpreferred GATCs sites 10 and 19 base pairs from either end of the DNA and 29 bp from each other, respectively.…”

Mechanistic insights into editing-site specificity of ADARs

“…However, definitive proof of this mechanism awaits further experimentation. Other mechanisms used by base-flipping enzymes include the serine-mediated pinch-pull-push mechanism (30) and the helixbending, base-flipping, and intercalation mechanism (26). An alternative passive mechanism also has been proposed in which the protein simply traps a transiently flipped-out base (31).…”

“…In M.EcoRI, an N6-adenine DNA methyl transferase that uses a bending, base-flipping, and intercalation mechanism, a bending-deficient mutant decreases base flipping and increases specificity (24,25). For noncognate substrates, M.EcoRI specificity arises from partitioning the enzyme/DNA intermediate into the unbent form (25,26). We investigated whether E488Q and T490A, mutants that showed differences in 2-AP FI compared with WT hADAR2, also showed differences in substrate specificity.…”

Nucleic Acid–Based Nanodevices in Biological Imaging

“…Precise positioning of the attachment sites of the dsDNA template on the origami framework can modulate the tension and bending of the displayed dsDNA substrate. Many DNA-modifying enzymes require that their dsDNA substrate possesses the capacity to bend (33). For example, methylation on adenine by EcoRI methyltransferase (M.EcoRI) occurs upon a 55–59° bending of dsDNA.…”