Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy
Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy
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DOI：
10.1006/meth.2001.1216
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发表时间：
2001-09-01
期刊：
影响因子：
4.8
通讯作者：
Millar, DP
中科院分区：
文献类型：
--
作者：
Bailey, MF;Thompson, EHZ;Millar, DP
在进行合成后3‘-5’编辑(校对)之前，必须将有缺陷的DNA引物末端从5‘-3’聚合酶活性部位转移到远程3‘-5’核酸外切酶部位。为了阐明发生这种情况的机制，我们使用时间分辨荧光光谱研究了丹磺酰标记的DNA引物/模板与大肠杆菌DNA聚合酶1的Klenow片段的相互作用。丹磺酰探针的位置使得当DNA底物占据聚合酶活性部位时，探针暴露在溶剂中，具有较短的平均荧光寿命(4.7 ns)和广泛的角扩散(42.5度)。相反，当DNA底物占据核酸外切酶活性部位时，探针被埋在蛋白质内部，导致平均寿命增加(14.1 ns)，角扩散程度降低(14.4度)。如果同时填充聚合酶和核酸外切酶结合模式(下限类似于5%)，它们明显不同的荧光性质会导致各向异性以典型的“下降和上升”的形式衰减。对这些数据的非线性最小二乘分析恢复了暴露的(x(E))和掩埋的(x(B))探针的基态摩尔分数，相当于DNA底物分别结合在聚合酶和核酸外切酶位点的平衡比例。聚合酶和核酸外切酶结合模式之间的分布由平衡分配常数K-pe(等于x(B)/x(E))给出。因此，校对过程的重要决定因素可以通过对蛋白质或DNA的改变来确定，这些改变扰乱了分配平衡，从而改变了K-pe的大小。(C)2001年学术出版社。
Prior to undergoing postsynthetic 3 ' -5 ' editing (proofreading), a defective DNA primer terminus must be transferred from the 5 ' -3 ' polymerase active site to a remote 3 ' -5 ' exonuclease site. To elucidate the mechanisms by which this occurs, we have used time-resolved fluorescence spectroscopy to study the interaction of dansyl-labeled DNA primer/templates with the Klenow fragment of Escherichia coli DNA polymerase 1. The dansyl probe is positioned such that when the DNA substrate occupies the polymerase active site, the probe is solvent-exposed and possesses a short average fluorescence lifetime (4.7 ns) and extensive angular diffusion (42.5 degrees). Conversely, when the DNA substrate occupies the exonuclease active site, the probe becomes buried within the protein, resulting in an Increase in the average lifetime (14.1 ns) and a decrease in the degree of angular diffusion (14.4 degrees). If both polymerase and exonuclease binding modes are populated (lower limit similar to5%), their markedly different fluorescence properties cause the anisotropy to decay with a characteristic "dip and rise" shape. Nonlinear least-squares analysis of these data recovers the ground-state mole fractions of exposed (x(e))and buried (x(b)) probes, which are equivalent to the equilibrium proportions of the DNA substrate bound at the polymerase and exonuclease sites, respectively. The distribution between the polymerase and exonuclease binding modes is given by the equilibrium partitioning constant K-pe (equal to x(b)/x(e)). The important determinants of the proofreading process can therefore be identified by changes made to either the protein or DNA that perturb the partitioning equilibrium and hence alter the magnitude of K-pe. (C) 2001 Academic Press.