2-Aminopurine Flipped into the Active Site of the Adenine-Specific DNA Methyltransferase M.TaqI:  Crystal Structures and Time-Resolved Fluorescence

Abstract

We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state: base flipping is accompanied by the loss of the very short ( approximately 50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by pi-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the proposed key residue for base flipping by M.TaqI, the target base partner thymine, is substituted by an abasic site analog. The corresponding cocrystal structure shows 2-aminopurine in the active site of M.TaqI, demonstrating that the partner thymine is not essential for base flipping. However, in this structure, a shift of the 3' neighbor of the target base into the vacancy left after base flipping is observed, apparently replicating a stabilizing role of the missing partner thymine. Time-resolved fluorescence and acrylamide quenching measurements of M.TaqI complexes in solution provide evidence for an alternative binding site for the extra-helical target base within M.TaqI and suggest that the partner thymine assists in delivering the target base into the active site.

Mechanistic insights into editing-site specificity of ADARs

“…The analog is used frequently to report on base flipping (23), including with hADAR2 (20), and in limited cases the observed changes in fluorescence have been correlated with a crystal structure with 2-AP in a flipped-out position (28). Although we cannot be certain our assays with 2-AP are measuring base flipping, this explanation seems to be the most likely.…”

Long-distance placement of substrate RNA by H/ACA proteins

“…The quantum yield of 2-AP is sensitive to its environment and exhibits strong quenching when AP is stacked (Rachofsky et al 2001;Serrano-Andres et al 2006). Thus, 2-AP has been extensively used to probe dynamics and kinetics of DNAenzyme and RNA-enzyme interactions (Holz et al 1998;Stivers 1998;Lenz et al 2007). The identity of this nucleotide is not conserved, and its substitution by 2-AP is expected to have minimal impact on substrate binding and pseudouridylation activities.…”

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

“…The enhancement of 2-AP fluorescence induced by DNAbinding proteins has been interpreted previously as evidence for DNA strand separation and/or base flipped out of its helical conformation. [42][43][44][45] Although the precise mechanism remains obscure, helical distortions may account for the fluorescence enhancement induced by PI-MleI. We conclude, however, that changes in DNA conformation are apparent at the cleavage sites upon binding of PI-MleI and its variants; the absence of such distortion at the binding DNA sequence (Fig.…”

“…41 Fluorescent base analogs, such as 2-AP, are often used to monitor such conformational changes involving proteinnucleic acid interaction. [42][43][44][45] To gain insights into the functional differences in the target DNA selectivity of wild-type PI-MleI and its variants, we used 2-AP fluorescence to monitor changes induced in double-helical DNA by these enzymes. We observed that the fluorescence intensities induced by PI-MleI and its variants with the target DNA substrate at the cleavage site followed a hierarchical manner in the following order: wild-type PI-MleI : D122C : D122T : D218A > D122A > D193A (Fig.…”