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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 ?M. 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.