Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion.
Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion.
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DOI：
10.1038/ncomms3131
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发表时间：
2013
影响因子：
16.6
通讯作者：
Kapanidis, Achillefs N.
中科院分区：
文献类型：
--
作者：
Hohlbein, Johannes;Aigrain, Louise;Craggs, Timothy D.;Bermek, Oya;Potapova, Olga;Shoolizadeh, Pouya;Grindley, Nigel D. F.;Joyce, Catherine M.;Kapanidis, Achillefs N.
DNA聚合酶的保真度取决于构象变化，这些构象变化促进在磷酰基转移之前排斥不正确的核苷酸。在这里，我们结合联合收割机单分子FRET与DNA聚合酶I和各种保真度突变体的使用，以突出的机制，活性位点侧链的影响构象转变和自由能景观，在DNA合成的保真度决定的基础。高保真衍生物与互补dNTPs的三元复合物主要采用完全闭合的构象，而FRET值介于开放和闭合构象之间的构象稀疏分布。这种中间FRET状态，我们归因于一个部分封闭的构象，也是占主导地位的三元复合物与不正确的核苷酸，引人注目的是，在大多数三元复合物的低保真度衍生物的正确和不正确的核苷酸。低保真度衍生物的突变表型与互补dNTPs的亲和力降低密切相关，并突出显示部分闭合的构象作为核苷酸选择的主要检查点。 DNA聚合酶的保真度取决于构象变化，这些构象变化促进了对不正确核苷酸的排斥。在这里，通过使用分子内单分子FRET测定，作者建立和表征部分闭合构象作为一个重要的保真度检查点。
The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection. The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides. Here, by using an intramolecular single-molecule FRET assay, the authors establish and characterize the partially closed conformation as a crucial fidelity checkpoint.
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影响因子：
3.3
作者：
Kalinin, Stanislav;Valeri, Alessandro;Seidel, Claus A. M.
通讯作者：
Seidel, Claus A. M.
DOI：
10.1073/pnas.032457899
发表时间：
2002-02-05
影响因子：
11.1
作者：
Minnick, DT;Liu, LX;Joyce, CM
通讯作者：
Joyce, CM
影响因子：
2.9
作者：
Joyce, Catherine M.;Potapova, Olga;Grindley, Nigel D. F.
通讯作者：
Grindley, Nigel D. F.
影响因子：
5.6
作者：
Astatke, M;Grindley, NDF;Joyce, CM
通讯作者：
Joyce, CM
影响因子：
7.4
作者：
Santoso, Yusdi;Kapanidis, Achillefs N.
通讯作者：
Kapanidis, Achillefs N.