Key Laboratory of Environment and Female Reproductive Health, West China School of Public Health & West China Fourth Hospital, Sichuan University, Chengdu, 610041, China.
College of Biological Sciences and Engineering, North Minzu University, Yinchuan, Ningxia, 750021, China.
Biochimie. 2019 Dec;167:25-33. doi: 10.1016/j.biochi.2019.09.002. Epub 2019 Sep 4.
rNTPs are structurally similar to dNTPs, but their concentrations are much higher than those of dNTPs in cells. rNTPs in solutions or rNMP at the primer terminus or embedded in template always inhibit or block DNA replication, due to the reduced Mg apparent concentration, competition of rNTPs with dNTPs, and the extra repulsive interaction of rNTP or rNMP with polymerase active site. In this work, unexpectedly, we found rNTPs can promote T7 DNA replication with the maximal promotion at rNTPs/dNTPs concentration ratio of 20. This promotion was not due to the optimized Mg apparent concentration or the direct incorporation of extra rNMPs into DNA. This promotion was dependent on the concentrations and types of rNTPs. Kinetic analysis showed that this promotion was originated from the increased fraction of polymerase-DNA productive complex and the accelerated DNA polymerization. Further evidence showed that more polymerase-DNA complex was formed and their binding affinity was also enhanced in the presence of extra rNTPs. Moreover, this promotion in T7 DNA replication also accelerated the lysis of T7-infected host Escherichia coli. This work discovered that rNTPs could promote DNA replication, completely different from the traditional concept that rNTPs always inhibit DNA replication.
rNTP 与 dNTP 在结构上相似，但它们在细胞中的浓度比 dNTP 高得多。溶液中的 rNTP 或引物末端的 rNMP 或嵌入模板中的 rNMP 总是由于降低的 Mg 表观浓度、rNTP 与 dNTP 的竞争以及 rNTP 或 rNMP 与聚合酶活性位点的额外排斥相互作用而抑制或阻断 DNA 复制。在这项工作中，出人意料的是，我们发现 rNTP 可以促进 T7 DNA 复制，在 rNTP/dNTP 浓度比为 20 时达到最大促进作用。这种促进作用不是由于优化的 Mg 表观浓度或额外 rNMP 直接掺入 DNA 所致。这种促进作用取决于 rNTP 的浓度和类型。动力学分析表明，这种促进作用源于聚合酶-DNA 有效复合物分数的增加和 DNA 聚合的加速。进一步的证据表明，在存在额外的 rNTP 时，更多的聚合酶-DNA 复合物形成，并且它们的结合亲和力也增强。此外，这种 T7 DNA 复制的促进作用也加速了 T7 感染宿主大肠杆菌的裂解。这项工作发现，rNTP 可以促进 DNA 复制，这与 rNTP 总是抑制 DNA 复制的传统观念完全不同。
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