Use of viscogens, dNTPαS, and rhodium(III) as probes in stopped-flow, experiments to obtain new evidence for the mechanism of catalysis by DNA polymerase β
Use of viscogens, dNTPαS, and rhodium(III) as probes in stopped-flow, experiments to obtain new evidence for the mechanism of catalysis by DNA polymerase β
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DOI：
10.1021/bi047664w
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发表时间：
2005-04-05
期刊：
影响因子：
2.9
通讯作者：
Tsai, MD
中科院分区：
文献类型：
--
作者：
Bakhtina, M;Lee, S;Tsai, MD
DNA聚合酶保真度的动力学机制和结构基础仍存在很大争议。在这里，我们报告使用三个探针在停止流动的研究Pol β，以获得新的，直接的证据，我们以前的解释：（a）增加反应缓冲液的粘度，蔗糖或甘油，预计减缓差异的构象变化，它被证明减缓第一（快）荧光转换选择性。(b)使用dNTP α S代替dNTP预期优先减慢化学步骤，并且显示选择性地减慢第二（慢）荧光跃迁。(c)使用置换惰性Rh（III）dNTP首次显示在Pol β中心点DNA中心点Rh（III）dNTP与Mg（II）混合后发生缓慢的荧光变化。这些结果，沿着与晶体结构，表明子域关闭构象变化发生在结合的催化Mg（II），而限速步骤发生后，结合的催化Mg（II）。这些结果为我们先前提出的机制提供了新的证据，但不支持最近三篇计算研究论文的结果。该结果进一步得到了“顺序混合”停流实验的支持，该实验没有使用类似物，因此排除了实验和计算结果之间的差异是由于使用类似物的可能性。该方法可用于检查其他DNA聚合酶，以回答Pol beta的性质是例外还是一般。
The kinetic mechanism and the structural bases of the fidelity of DNA polymerases are still highly controversial. Here we report the use of three probes in the stopped-flow studies of Pol beta to obtain new, direct evidence for our previous interpretations: (a) Increasing the viscosity of the reaction buffer by sucrose or glycerol is expected to slow down the conformational change differentially, and it was shown to slow down the first (fast) fluorescence transition selectively. (b) Use of dNTP alpha S in place of dNTP is expected to slow down the chemical step preferentially, and it was shown to slow down the second (slow) fluorescence transition selectively. (c) The substitution-inert Rh(III)dNTP was used to show for the first time that the slow fluorescence change occurs after mixing of Pol beta center dot DNA center dot Rh(III)dNTP with Mg(II). These results, along with crystal structures, suggest that the subdomain-closing conformational change occurs before binding of the catalytic Mg(II) while the rate-limiting step occurs after binding of the catalytic Mg(II). These results provide new evidence to the mechanism we suggested previously, but do not support the results of three recent papers of computational studies. The results were further supported by a "sequential mixing" stopped-flow experiment that used no analogues, and thus ruled out the possibility that the discrepancy between experimental and computational results is due to the use of analogues. The methodologies can be used to examine other DNA polymerases to answer whether the properties of Pol beta are exceptional or general.