DNA Bending by EcoRI DNA Methyltransferase Accelerates Base Flipping but Compromises Specificity

Abstract

EcoRI DNA methyltransferase was previously shown to bend its cognate DNA sequence by 52°and stabilize the target adenine in an extrahelical orientation. We describe the characterization of an EcoRI DNA methyltransferase mutant in which histidine 235 was selectively replaced with asparagine. Steady-state kinetic and thermodynamic parameters for the H235N mutant revealed only minor functional consequences: DNA binding affinity (K D DNA ) was reduced 10-fold, and k cat was decreased 30%. However, in direct contrast to the wild type enzyme, DNA bending within the mutant enzyme-DNA complexes was not observed by scanning force microscopy. The bending-deficient mutant showed enhanced discrimination against the methylation at nontarget sequence DNA. This enhancement of enzyme discrimination was accompanied by a change in the rate-limiting catalytic step. No presteady-state burst of product formation was observed, indicating that the chemistry step (or prior event) had become rate-limiting for methylation. Direct observation of the base flipping transition showed that the lack of burst kinetics was entirely due to slower base flipping. The combined data show that DNA bending contributes to the correct assembly of the enzyme-DNA complex to accelerate base flipping and that slowing the rate of this precatalytic isomerization can enhance specificity.The formation of protein-DNA complexes frequently requires distortion of the DNA from a B-conformation (1). Recently, the unstacking and subsequent extrahelical stabilization of a DNA base ("base flipping") has expanded the examples of enzymemediated DNA deformations. Extrahelical base stabilization enables access of the catalytic enzyme groups to inaccessible target DNA residues, providing an elegant mechanism whereby discrimination can be achieved. For S-adenosyl-L-methioninedependent DNA methyltransferases, base flipping also allows the modification of a large number of DNA sequences without the stereochemical constraint of cofactor-DNA interactions.X-ray crystal structures reveal that the C 5 cytosine-specific type II bacterial DNA methyltransferases HaeIII DNA cytosine methyltransferase and HhaI DNA cytosine methyltransferase stabilize an extrahelical base without significantly bending DNA (2, 3). Similarly, uracil DNA glycosylase stabilizes an extrahelical uracil without further distortion of the DNA configuration (4). In contrast, T4 endonuclease V recognizes thymine dimers, flips an adenine opposite to the lesion, and also bends the DNA by 60°(5). Functional studies of the N 6 -adenine EcoRI DNA methyltransferase (M.EcoRI) 1 and the related EcoRV DNA adenine methyltransferase show that these adenine-specific base flipping enzymes also bend DNA (6, 7).The enzyme-DNA cocrystal structures provide compelling evidence for the stabilization of distorted DNA conformations; yet, surprisingly little is known concerning the coordination between sequence-specific DNA base recognition and flipping. Determining the temporal coordination between the binding of the enzyme to the DNA an...

Observing an Induced-fit Mechanism during Sequence-specific DNA Methylation

“…Third, our recent work on M.HhaI and other DNA methyltransferases suggests that conformational rearrangements such as the loop movement in M.HhaI or DNA bending by M.EcoRI can directly contribute to specificity (29,30,35). Here, we extend our prior studies (29,30) by directly probing loop motion using the fluorescence signal of tryptophans engineered into the flexible loop (80 -100). Importantly, the highly conserved nature of this loop among DNA cytosine methyltransferases (36) suggests our results may be broadly applicable.…”

“…Second, the temporal assignment of loop movement in relation to other known steps in the reaction cycle, such as base flipping, could be insightful for understanding if these processes are enzyme-assisted or occur passively (9,34). Third, our recent work on M.HhaI and other DNA methyltransferases suggests that conformational rearrangements such as the loop movement in M.HhaI or DNA bending by M.EcoRI can directly contribute to specificity (29,30,35). Here, we extend our prior studies (29,30) by directly probing loop motion using the fluorescence signal of tryptophans engineered into the flexible loop (80 -100).…”

Simultaneous DNA Binding, Bending, and Base Flipping

“…Similar base flipping conformational transitions have now been shown for diverse DNA methyltransferases, DNA repair enzymes, and RNA-modifying enzymes (13). Some DNA methyltransferases also bend their cognate DNA sequence by 50 -90°as part of their recognition mechanism, and the bending and base flipping process are energetically coupled (14,15).…”

“…Scheme I summarizes these characterized transitions and kinetic constants for M.EcoRI. Recently, we described an intriguing M.EcoRI mutant, H235N, which does not appear to bend its cognate sequence, yet shows largely wild-type steady state parameters (15). Surprisingly, this bending-deficient mutant sequence-discrimination is enhanced at least 1000-fold, suggesting a close relationship between DNA bending and specificity.…”

“…The bacterial DNA adenine methyltransferase M.EcoRI modifies the second adenine in the palindromic sequence GAATTC (15). The enzyme uses a facilitated diffusion mechanism to locate its sites within the bacterial genome, and its specificity constant (k cat /K m ) is near diffusion-limited (16,17).…”