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

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 (∼50 ps) lifetime component associated with fully base-stacked
2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by π-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.