Jia Xiaomeng, Gao Xiang, Zhang Shuming, Inman James T, Hong Yifeng, Singh Anupam, Patel Smita, Wang Michelle D
Howard Hughes Medical Institute, Cornell University, Ithaca, NY 14853, USA.
Department of Physics & LASSP, Cornell University, Ithaca, NY 14853, USA.
bioRxiv. 2024 Oct 17:2024.10.14.618227. doi: 10.1101/2024.10.14.618227.
The inherent helical structure of DNA dictates that a replisome must rotate relative to DNA during replication, presenting inevitable topological challenges to replication. However, little is known about how the replisome progresses against torsional stress. Here, we developed a label-free, high-resolution, real-time assay to monitor replisome movement under torsion. We visualized the replisome rotation of DNA and determined how the replisome slows down under torsion. We found that while helicase or DNA polymerase (DNAP) individually is a weak torsional motor, the replisome composed of both enzymes is the most powerful DNA torsional motor studied to date. It generates ~ 22 pN·nm of torque before stalling, twice the stall torque of RNA polymerase. Upon replisome stalling, the specific interaction between helicase and DNAP stabilizes the fork junction; without it, the fork can regress hundreds of base pairs. We also discovered that prolonged torsion-induced stalling inactivates the replisome. Surprisingly, DNAP exchange, mediated by the helicase, is highly effective in facilitating replication restart, but only if excess DNAP is present during stalling. Thus, helicase and DNA polymerase work synergistically as a powerful torsional motor, and their dynamic and fluid interactions are crucial for maintaining fork integrity under torsional stress. This work demonstrates that torsion is a strong regulator of DNA replication stalling and reactivation.
DNA的固有螺旋结构决定了在复制过程中，复制体必须相对于DNA发生旋转，这给复制带来了不可避免的拓扑学挑战。然而，关于复制体如何克服扭转应力进行复制，我们所知甚少。在这里，我们开发了一种无标记、高分辨率的实时检测方法，以监测扭转条件下复制体的运动。我们可视化了DNA的复制体旋转，并确定了复制体在扭转条件下是如何减速的。我们发现，虽然解旋酶或DNA聚合酶（DNAP）单独作为扭转马达的能力较弱，但由这两种酶组成的复制体是迄今为止研究的最强大的DNA扭转马达。它在停滞前产生约22皮牛·纳米的扭矩，是RNA聚合酶停滞扭矩的两倍。复制体停滞时，解旋酶与DNAP之间的特异性相互作用稳定了叉形连接；没有这种相互作用，叉形结构可能会倒退数百个碱基对。我们还发现，长时间的扭转诱导停滞会使复制体失活。令人惊讶的是，由解旋酶介导的DNAP交换在促进复制重新启动方面非常有效，但前提是在停滞期间存在过量的DNAP。因此，解旋酶和DNA聚合酶协同作用，作为一个强大的扭转马达，它们动态而灵活的相互作用对于在扭转应力下维持叉形结构的完整性至关重要。这项工作表明，扭转是DNA复制停滞和重新激活的一个强有力的调节因子。
