Affinity photo-crosslinking study of the DNA base flipping pathway by HhaI methyltransferase

HhaI DNA methyltransferase flips the inner cytosine in the recognition sequence 5'-GCGC-3' out of the DNA helix and into the catalytic site for methylation. To identify intermediate states on the base-flipping pathway, affinity photo-crosslinking experiments were performed with synthetic dodecamer duplexes containing modified bases 2-thiothymine (2sT) or 4-thiothymine (4sT) at the target base position. Here we show that the DNA strand containing 2sT, but not 4sT, covalently cross-links to the HhaI methyltransferase upon irradiation at 340-360 nm.