Simultaneous DNA Binding, Bending, and Base Flipping

Abstract

We measured the kinetics of DNA bending by M.EcoRI using DNA labeled at both 5-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3-ends, the effect is cofactor-and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from M.EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex. The M.EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly, M.EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes. Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.Structural transitions involving substrate-induced changes in enzyme conformation and protein-induced changes in substrate conformation occur in diverse classes of enzymes. The quantitative importance of such mechanisms toward catalysis and specificity has been demonstrated for DNA polymerases (1), phytase (2), integration host factor (3), pyridoxal kinase (4), restriction enzymes, DNA repair enzymes (5), and DNA modifying enzymes (6). Yet, conformational mechanisms are difficult to study because the reaction intermediates are largely inaccessible to classical kinetic analysis. Spectroscopic probes and, in particular, the highly distant-dependent method of fluorescence resonance energy transfer (FRET) 1 provide a viable solution-based approach to characterize conformational intermediates (7). Only through the quantitative analysis of stable conformational intermediates and the kinetic transitions involving their interconversion can a mechanistic understanding of catalysis and specificity be approached.DNA methyltransferases provide a rich system to investigate the mechanisms and importance of conformational transitions. The enzymes are structurally characterized, carry out both DNA bending and base flipping (8), and the bacterial and human enzymes are the targets of novel antibiotic (9) and cancer drug development efforts respectively (10). These l...

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).…”

Conformational Transitions as Determinants of Specificity for the DNA Methyltransferase EcoRI

“…Cognate duplex DNA is defined as cognate bottom methylated. Oligonucleotides for FRET studies were coupled as described previously (18). Briefly, oligonucleotides were resuspended in distilled H 2 O, extracted with chloroform, ethanol-precipitated, and resuspended to a final concentration of 25 g/l.…”

“…Enzyme Expression and Purification-M.EcoRI was purified from MM294 Escherichia coli cells with the plasmid pXRI as described previously (18). 6 liters of culture were induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside at an A 600 0.4 for 3 h. Cells were sonicated in extraction buffer (200 mM NaCl, 6.5 mM K 2 HPO 4 , 3.5 mM KH 2 PO 4 , 1 mM EDTA, and protease inhibitor mixture (Sigma)).…”

“…Upon location of the target sequence, M.EcoRI induces a 50°bend in the DNA sequence and flips the target adenine out 180°to an extrahelical position to facilitate catalysis. Solution-based FRET 2 studies were used to demonstrate that along with bending the DNA, M.EcoRI also expands the duplex DNA structure presumably through an intercalative mechanism (18), and intercalation and bending occur nearly simultaneously with DNA binding at 25°C (18).…”