Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity
Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity
复制标题
DOI：
10.1016/j.bpj.2016.12.044
复制
发表时间：
2017-02-28
影响因子：
3.4
通讯作者：
Wuite, Gijs J. L.
中科院分区：
文献类型：
--
作者：
Hoekstra, Tjalle P.;Depken, Martin;Wuite, Gijs J. L.
DNA聚合酶催化遗传信息从一代到下一代的准确传递，因此复制的忠实性至关重要。DNA聚合酶通过以下机制的组合满足对保真度的严格要求：1）对正确核苷酸掺入的高选择性，2）在错误掺入后减慢复制速率，和3）通过切除错误掺入的碱基来校正。为了阐明复制和校对之间的动力学相互作用，我们使用高分辨率光镊探测DNA双链体稳定性如何影响噬菌体T7 DNA聚合酶的复制。我们的数据显示了高度不规则的复制动态，随着聚合酶循环通过控制高保真T7 DNA复制背后的机械化学的状态，频繁的停顿和方向逆转。我们构建了一个动力学模型，该模型结合了现有的生化数据和据我们所知，我们观察到的新状态。我们将模型直接拟合到获得的暂停时和运行时分布。我们的研究结果表明，错误纠正的主要途径是DNA聚合酶解离介导的DNA转移，其次是偏向结合到核酸外切酶的活性位点。通过这种校对机制去除的碱基数量远大于预期将被并入的错误碱基的数量，从而确保噬菌体T7基因组的高保真复制。
DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome.