DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase
DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase
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DOI：
10.1073/pnas.1012277107
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发表时间：
2010-10-19
影响因子：
11.1
通讯作者：
von Hippel, Peter H.
中科院分区：
文献类型：
--
作者：
Datta, Kausiki;Johnson, Neil P.;von Hippel, Peter H.
引物-模板（P/T）DNA中的局部构象变化参与DNA聚合酶（DNAP）对dNTP的选择性掺入。在这里，我们使用在引物末端或模板链编码区取代的碱基类似物探针对的近紫外CD和荧光光谱来监测和解释P/T DNA复合物中模板编码碱基及其附近的构象变化。随着聚合酶在核苷酸添加循环中移动，Klenow片段（KF）DNAP。进入的dNTP和rNTP遇到二元复合物，其中引物的3 '末端在DNAP的聚合（pol）和核酸外切酶（exo）位点之间穿梭，即使对于完全互补的P/T DNA序列也是如此。我们已经使用光谱变化的探针插入到两条链来监测这种两种状态的分布，并确定它如何依赖于形成三元复合物与互补（“正确”）和非互补（“不正确”）的NTPs和本地序列的P/T DNA。结果表明，exo和pol位点的相对占据率与复合物的P/T DNA中的构象变化相关联，所述构象变化部分地由进入的NTP调节。我们发现模板链上的编码碱基不受不正确的dNTPs结合的干扰，而互补的rNTPs的结合诱导了一种新的模板构象。我们的结论是，除了它的编辑功能，引物链占据的3 '-外切位点也可以作为一个监管检查点，在DNA合成中准确的dNTP选择。
Local conformational changes in primer-template (P/T) DNA are involved in the selective incorporation of dNTP by DNA polymerases (DNAP). Here we use near UV CD and fluorescence spectra of pairs of base analogue probes, substituted either at the primer terminus or in the coding region of the template strand, to monitor and interpret conformational changes at and near the coding base of the template in P/T DNA complexes with Klenow fragment (KF) DNAP as the polymerase moves through the nucleotide addition cycle. Incoming dNTPs and rNTPs encounter binary complexes in which the 3'-end of the primer shuttles between the polymerization (pol) and exonuclease (exo) sites of DNAPs, even for perfectly complementary P/T DNA sequences. We have used spectral changes of probes inserted in both strands to monitor this two-state distribution and determine how it depends on the formation of ternary complexes with both complementary ("correct") and noncomplementary ("incorrect") NTPs and on the local sequence of the P/T DNA. The results show that the relative occupancy of the exo and pol sites is coupled to conformational changes in the P/T DNA of the complex that are partially regulated by the incoming NTP. We find that the coding base on the template strand is unperturbed by the binding of incorrect dNTPs, while binding of complementary rNTPs induces a novel template conformation. We conclude that, in addition to its editing function, primer strand occupancy of the 3'-exo site may also serve as a regulatory checkpoint for accurate dNTP selection in DNA synthesis.