Direct Real Time Observation of Base Flipping by theEcoRI DNA Methyltransferase

Abstract

DNA methyltransferases are excellent prototypes for investigating DNA distortion and enzyme specificity because catalysis requires the extrahelical stabilization of the target base within the enzyme active site. The energetics and kinetics of base flipping by the EcoRI DNA methyltransferase were investigated by two methods. First, equilibrium dissociation constants (K D DNA ) were determined for the binding of the methyltransferase to DNA containing abasic sites or base analogs incorporated at the target base. Consistent with a base flipping mechanism, tighter binding to oligonucleotides containing destabilized target base pairs was observed. Second, total intensity stopped flow fluorescence measurements of DNA containing 2-aminopurine allowed presteadystate real time observation of the base flipping transition. Following the rapid formation of an enzyme-DNA collision complex, a biphasic increase in total intensity was observed. The fast phase dominated the total intensity increase with a rate nearly identical to k methylation determined by rapid chemical quench-flow techniques (Reich, N. O., and Mashoon, N. (1993) J. Biol. Chem. 268, 9191-9193). The restacking of the extrahelical base also revealed biphasic kinetics with the recovered amplitudes from these off-rate experiments matching very closely to those observed during the base unstacking process. These results provide the first direct and continuous observation of base flipping and show that at least two distinct conformational transitions occurred at the flipped base subsequent to complex formation. Furthermore, our results suggest that the commitment to catalysis during the methylation of the target site is not determined at the level of the chemistry step but rather is mediated by prior intramolecular isomerization within the enzyme-DNA complex.Protein-DNA complexes reveal diverse mechanisms leading to sequence-specific interaction. Direct readout of DNA base functionalities within the major groove and the indirect readout of sequence-dependent phosphate backbone geometry are thought to contribute binding discrimination (1, 2). For DNA modification and repair enzymes the correct assembly of active site residues frequently demands the insertion of protein side chains into and rotating of a base completely out of the DNA helix (3, 4). The stabilization of an extrahelical base is often coupled to sequence-dependent DNA base pair rearrangement (5) and DNA bending (6). However, the energetic cost of the enzyme-mediated DNA deformations integrating site-specific recognition and catalysis are only now being elucidated.The mechanism leading to the stabilization of an extrahelical base is thought to involve a multi-step binding process with discrete conformational intermediates (4, 7). Enzyme-mediated weakening or breakage of Watson-Crick hydrogen bonds at the target base pair and intercalation of amino acid side chains into the DNA helix are likely to be critical to the initiation of the base flipping process (8). The enhanced discrimination provided by the maj...

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

“…Thus, the disruption of the internal hydrogen bonds between the cytosine and the base to which it is paired does not contribute to the observed k chem rates. This is in contrast to our previous observations showing that the baseflipping transition limits the single-turnover methylation kinetics for the adenine methyltransferase M.EcoRI (49). Since base flipping a cytosine from a guanosine:cytosine base pair is expected to be more difficult than an adenosine from a thymine:adenosine base pair due to the extra hydrogen bond (50), one would expect k chem measurements for cytosine methyltransferases to also be dominated by the base-flipping transition.…”

Photophysical Characterization of Fluorescent DNA Base Analogue, tC

“…polymerases, [8][9][10][11][12] restriction endonucleases, [13][14][15] and repair enzymes. [16][17][18][19] However, the sensitivity of its excited state lifetimes to environment makes it rather unreliable as a probe of molecular dynamics using fluorescence anisotropy measurements and energy transfer.…”

Fluorescence intensity decays of 2-aminopurine solutions: Lifetime distribution approach

“…Third, fluorescence emission from 2AP strongly depends on local environment [7,8] and is quenched by neighboring bases in DNA/RNA. 2AP has been successfully used to study interaction of DNA and RNA with proteins [9][10][11][12][13], repair enzymes [14][15][16], folding of ribozymes [17], DNA damage and mismatching [18,19].…”