title: Fluorescent Base Analogue Reveals T-HgII-T Base Pairs Have High Kinetic Stabilities That Perturb DNA Metabolism.
abstract: The thymidine analogueDMAT was used for the first fluorescence-based study of direct, site-specific metal binding reactions involving unmodified nucleobases in duplex DNA. The fluorescence properties ofDMAT-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-HgII-T binding reactions. The on- and off-rates of HgIIwere surprisingly slow, with association rate constants (kon) ≈ 104-105M-1s-1, and dissociation rate constants (koff) ≈ 10-4-10-3s-1; giving equilibrium dissociation constants (Kd) = 8-50 nM. In contrast, duplexes lacking a T-T mismatch exhibited local, nonspecific HgIIbinding affinities in the range of Kd= 0.2-2.0 μM, depending on the buffer conditions. The exceptionally high kinetic stabilities of T-HgII-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-HgII-T binding reactions in duplex DNA, these results demonstrate that T-HgII-T base pairs have a high potential to disrupt DNA metabolism in vivo.
authors.affiliation: Department of Chemistry, University of Zürich , Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Olivia P
authors.initials: OP
authors.lastname: Schmidt
authors.affiliation: Department of Chemistry, University of Zürich , Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Mata
authors.affiliation: Department of Chemistry, University of Zürich , Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Luedtke
mesh_terms: D020029:Base Pairing; D004247:DNA / Q000737:chemistry*; D007700:Kinetics; D008628:Mercury / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D013816:Thermodynamics; D013941:Thymine / Q000737:chemistry*
chemical_list: D004247:DNA; D008628:Mercury; D013941:Thymine