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A fluorescent surrogate of thymidine called DMAT was used for the first fluorescence-based study of HgII binding to discrete T-T sites in duplex DNA. The fluorescent properties of DMAT-A base pairs were highly sensitive to wild-type T-HgII-T base pair formation at an adjacent site, allowing for a determination of the precise thermodynamic and kinetic parameters of these metal binding reactions. T-HgII-T complexes exhibited equilibrium dissociation constants of Kd ≈ 8-50 nM. These high-affinity binding interactions are characterized by very slow association and dissociation kinetics (kon ≈ 104- 105 M-1s-1, koff ≈ 10-4 - 10-3s-1), revealing exceptionally high kinetic stabilities of T-HgII-T base pairs (half-lives = 0.3-1.3 h). Duplex DNA containing DMAT and no T-T mismatch exhibited nonspecific HgII binding affinities of Kd ≈ 2.0 μM. The high kinetic stabilities of T-HgII-T resulted in the inhibition of dynamic processes such as DNA strand invasion and strand displacement during enzymatic DNA synthesis, which led to premature chain termination. These results demonstrated that T-HgII-T base pairs are kinetically distinct from T-A base pairs and therefore are likely to disrupt DNA metabolism in vivo.

The thymidine analogue DMA T was used for the first fluorescence-based study of direct, site-specific metal binding reactions involving unmodified nucleobases in duplex DNA. The fluorescence properties of DMA T-A base pairs were highly sensitive to mercury binding reactions at T-T mismatches located at an adjacent site or one base pair away. This allowed for precise determination of the local kinetic and thermodynamic parameters of T-Hg II -T binding reactions. The on- and off-rates of Hg II were surprisingly slow, with association rate constants (k on ) ≈ 10 4 -10 5 M -1 s -1 , and dissociation rate constants (k off ) ≈ 10 -4 -10 -3 s -1 ; giving equilibrium dissociation constants (K d ) = 8-50 nM. In contrast, duplexes lacking a T-T mismatch exhibited local, nonspecific Hg II binding affinities in the range of K d = 0.2-2.0 μM, depending on the buffer conditions. The exceptionally high kinetic stabilities of T-Hg II -T metallo-base pairs (half-lives = 0.3-1.3 h) perturbed dynamic processes including DNA strand displacement and primer extension by DNA polymerases that resulted in premature chain termination of DNA synthesis. In addition to providing the first detailed kinetic and thermodynamic parameters of site-specific T-Hg II -T binding reactions in duplex DNA, these results demonstrate that T-Hg II -T base pairs have a high potential to disrupt DNA metabolism in vivo.