Using the fluorescence decay of 2-aminopurine to investigate conformational change in the recognition sequence of the EcoRV DNA-(adenine-N6)-methyltransferase on enzyme binding

The EcoRV DNA methyltransferase methylates the first adenine in the GATATC recognition sequence. It is presumed that methylation proceeds by a nucleotide flipping mechanism but no crystal structure is available to confirm this. A popular solution-phase assay for nucleotide flipping employs the fluorescent adenine analogue, 2-aminopurine (2AP), substituted at the methylation target site; a substantial increase in fluorescence intensity on enzyme binding indicates flipping. However, this appeared to fail for M.EcoRV, since 2AP substituted for the non-target adenine in the recognition sequence showed a much greater intensity increase than 2AP at the target site. This anomaly is resolved by recording the fluorescence decay of 2AP which shows that the target 2AP is indeed flipped by the enzyme, but its fluorescence is quenched by interaction with aromatic residues in the catalytic site, whereas bending of the duplex at the non-target site alleviates inter-base quenching and exposes the 2AP to solvent.

Highlights► The anomalous response of 2AP intensity to M.EcoRV binding is resolved by measuring the fluorescence decay. ► The fluorescence decay parameters of 2AP report unambiguously and informatively on DNA conformation and the DNA-enzyme interface. ► When M.EcoRV binds to its recognition sequence it induces different local distortions at the two adenine positions. ► The target 2AP is flipped, but its fluorescence is quenched by interaction with aromatic residues in the catalytic site. ► Bending of the duplex at the non-target site alleviates inter-base quenching and exposes the 2AP to solvent.