Key Laboratory of Environment and Female Reproductive Health, West China School of Public Health & West China Fourth Hospital, Sichuan University, Chengdu, China.
National Laboratory for Condensed Matter Physics and Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.
FASEB J. 2021 May;35(5):e21607. doi: 10.1096/fj.202100033RR.
Strand displacement DNA synthesis (SDDS) is an essential step in DNA replication. With magnetic tweezers, we investigated SDDS kinetics of wild-type gp90 and its exonuclease-deficient polymerase gp90 exo at single-molecule level. A novel binding state of gp90 to the fork flap was confirmed prior to SDDS, suggesting an intermediate in the initiation of SDDS. The rate and processivity of SDDS by gp90 exo or wt-gp90 are increased with force and dNTP concentration. The rate and processivity of exonuclease by wt-gp90 are decreased with force. High GC content decreases SDDS and exonuclease processivity but increases exonuclease rate for wt-gp90. The high force and dNTP concentration and low GC content facilitate the successive SDDS but retard the successive exonuclease for wt-gp90. Furthermore, increasing GC content accelerates the transition from SDDS or exonuclease to exonuclease. This work reveals the kinetics of SDDS in detail and offers a broader cognition on the regulation of various factors on SDDS at single-polymerase level.
链位移 DNA 合成（SDDS）是 DNA 复制的一个基本步骤。我们使用磁镊在单分子水平上研究了野生型 gp90 和其缺乏外切酶的聚合酶 gp90 exo 的 SDDS 动力学。在 SDDS 之前，确认了 gp90 与叉形瓣之间的一种新的结合状态，这表明在 SDDS 的起始过程中有一个中间状态。gp90 exo 或 wt-gp90 的 SDDS 速率和持续性随着力和 dNTP 浓度的增加而增加。wt-gp90 的外切酶活性的速率和持续性随着力的增加而降低。高 GC 含量降低了 SDDS 和外切酶的持续性，但增加了 wt-gp90 的外切酶活性。高的力和 dNTP 浓度以及低的 GC 含量有利于 wt-gp90 的连续 SDDS，但会延迟连续的外切酶反应。此外，增加 GC 含量会加速从 SDDS 或外切酶到外切酶的转变。这项工作详细揭示了 SDDS 的动力学，并在单聚合酶水平上提供了对各种因素对 SDDS 调节的更广泛认识。
