荧光碱基类似物揭示 T-HgII-T 碱基对具有干扰 DNA 代谢的高动力学稳定性,Journal of the American Chemical Society

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荧光碱基类似物揭示 T-HgII-T 碱基对具有干扰 DNA 代谢的高动力学稳定性
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2016-10-25 , DOI: 10.1021/jacs.6b09044
Olivia P. Schmidt ₁ , Guillaume Mata ₁ , Nathan W. Luedtke ₁

Affiliation

胸苷类似物 DMAT 用于第一次基于荧光的直接、位点特异性金属结合反应研究，涉及双链 DNA 中未修饰的核碱基。DMAT-A 碱基对的荧光特性对位于相邻位点或一个碱基对处的 TT 错配处的汞结合反应高度敏感。这允许精确确定 T-HgII-T 结合反应的局部动力学和热力学参数。HgII 的开启和关闭速率出奇地慢，结合速率常数 (kon) ≈ 104-105 M-1 s-1，解离速率常数 (koff) ≈ 10-4-10-3 s-1；给出平衡解离常数 (Kd) = 8-50 nM。相比之下，缺乏 TT 错配的双链体在 Kd = 0.2-2.0 μM 范围内表现出局部非特异性 HgII 结合亲和力，具体取决于缓冲液条件。T-HgII-T 金属碱基对的极高动力学稳定性（半衰期 = 0.3-1.3 小时）扰乱了动态过程，包括 DNA 链置换和 DNA 聚合酶引物延伸，导致 DNA 合成链过早终止。除了提供双链 DNA 中位点特异性 T-HgII-T 结合反应的第一个详细动力学和热力学参数外，这些结果还表明 T-HgII-T 碱基对具有破坏体内 DNA 代谢的巨大潜力。

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Fluorescent Base Analogue Reveals T-HgII-T Base Pairs Have High Kinetic Stabilities That Perturb DNA Metabolism

The thymidine analogue DMAT 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 DMAT-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 HgII were surprisingly slow, with association rate constants (kon) ≈ 104-105 M-1 s-1, and dissociation rate constants (koff) ≈ 10-4-10-3 s-1; giving equilibrium dissociation constants (Kd) = 8-50 nM. In contrast, duplexes lacking a T-T mismatch exhibited local, nonspecific HgII binding 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.

更新日期：2016-10-25

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