Fluorescent Base Analogue Reveals T-HgII-T Base Pairs Have High Kinetic Stabilities That Perturb DNA Metabolism
Year: 2016
Type: article
Abstract: 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.
DOI: 10.1021/jacs.6b09044
Institution: University of Zurich
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2529027828