5-Fluorocytosine in DNA is a mechanism-based inhibitor of HhaI methylase

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Abstract
5-Fluorodeoxycytidine (FdCyd) was incorporated into a synthetic DNA polymer containing the GCGC recognition sequence of HhaI methylase to give a polymer with about 80% FdCyd. In the absence of AdoMet, poly(FdC-dG) bound competitively with respect to poly(dG-dC) (Ki = 3 nM). In the presence of AdoMet, the analogue caused a time-dependent, first-order (k = 0.05 min-1) inactivation of the enzyme. There is an ordered mechanism of binding in which enzyme first binds to poly(FdC-dG), then binds to AdoMet, and subsequently forms stable, inactive complexes. The complexes did not dissociate over the course of 3 days and were stable to heat (95.degree.C) in the presence of 1% SDS. Gel filtration of a complex formed with HhaI methylase, poly(FdC-dG), and [methyl-3H]AdoMet gave a peak of radioactivity eluting near the void volume. Digestion of the DNA in the complex resulted in a reduction of the molecular weight to the size of the methylase, and the radioactivity in this peak was shown to be associated with protein. These data indicate that the complexes contain covalently bound HhaI methylase, poly(FdC-dG), and methyl groups and that 5-fluorodeoxycytidine is a mechanism-based inactivator of the methylase. By analogy with other pyrimidine-modifying enzymes and recent studies on the mechanisms of HhaI methylase (Wu and Santi, 1987), these results suggest that an enzyme nucleophile attacks FdCyd residues at C-6, activating the 5-position for one-carbon transfer. Subsequent transfer of the methyl group of AdoMet to the activated FdCyd forms a stable complex in which the enzyme is covalently bound to the 6-position of FdCyd in the polymer and a methyl group is attached to C-5. The effect of 5-fluorodeoxycytidine on the inhibition of DNA-cytosine methyltransferases is thus due to irreversible, covalent inactivation.