Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase.

The methyltransferase, M.EcoKI, recognizes the DNA sequence 5'-AACNNNNNNGTGC-3' and methylates adenine at the underlined positions. DNA methylation has been shown by crystallography to occur via a base flipping mechanism and is believed to be a general mechanism for all methyltransferases. If no structure is available, the fluorescence of 2-aminopurine is often used as a signal for base flipping as it shows enhanced fluorescence when its environment is perturbed. We find that 2-aminopurine gives enhanced fluorescence emission not only when it is placed at the M.EcoKI methylation sites but also at a location adjacent to the target adenine. Thus it appears that 2-aminopurine fluorescence intensity is not a clear indicator of base flipping but is a more general measure of DNA distortion. Upon addition of the cofactor S-adenosyl-methionine to the M.EcoKI:DNA complex, the 2-aminopurine fluorescence changes to that of a new species showing excitation at 345 nm and emission at 450 nm. This change requires a fully active enzyme, the correct cofactor and the 2-aminopurine located at the methylation site. However, the new fluorescent species is not a covalently modified form of 2-aminopurine and we suggest that it represents a hitherto undetected physicochemical form of 2-aminopurine.

Resolution of racemic carbocyclic analogues of purine nucleosides through the action of adenosine deaminase. Antiviral activity of the carbocyclic 2'-deoxyguanosine enantiomers.

The action of adenosine deaminase on racemic carbocyclic analogues of 6-aminopurine nucleosides was investigated. When either racemic carbocyclic adenosine [(+/-)-C-Ado] or the racemic carbocyclic analogue [(+/-)-C-2,6-DAP-2'-dR] of 2,6-diaminopurine 2'-deoxyribofuranoside was incubated with this enzyme, approximately half of the material was deaminated rapidly. From the resulting solution, the D isomers of the deaminated carbocyclic analogues (D-carbocyclic inosine, D-C-Ino, or D-carbocyclic 2'-deoxyguanosine, D-2'-CDG) and the L isomers of the undeaminated carbocyclic analogues were isolated. At higher concentrations of the enzyme, deamination of L-C-Ado and L-C-2,6-DAP-2'-dR proceeded slowly, thus also making the other enantiomers accessible. In tests in vitro against herpes simplex virus, types 1 and 2, D-2'-CDG was as active and potent as (+/-)-2'-CDG, whereas L-2'-CDG displayed only modest activity. In contrast to the previously reported high activity and potency of (+/-)-C-2,6-DAP-2'-dR against these two viruses, L-C-2,6-DAP-2'-dR was inactive.