Kay Taryn M, Inman James T, Lubkowska Lucyna, Le Tung T, Qian Jin, Hall Porter M, Wang Dong, Kashlev Mikhail, Wang Michelle D
Biophysics Program, Cornell University, Ithaca, NY 14853, USA.
Department of Physics & LASSP, Cornell University, Ithaca, NY 14853, USA.
bioRxiv. 2024 Oct 13:2024.10.11.617674. doi: 10.1101/2024.10.11.617674.
DNA replication and transcription occur simultaneously on the same DNA template, leading to inevitable conflicts between the replisome and RNA polymerase. These conflicts can stall the replication fork and threaten genome stability. Although numerous studies show that head-on conflicts are more detrimental and more prone to promoting R-loop formation than co-directional conflicts, the fundamental cause for the RNA polymerase roadblock polarity remains unclear, and the structure of these R-loops is speculative. In this work, we use a simple model system to address this complex question by examining the Pol II roadblock to a DNA fork advanced via mechanical unzipping to mimic the replisome progression. We found that the Pol II binds more stably to resist removal in the head-on configuration, even with minimal transcript size, demonstrating that the Pol II roadblock has an inherent polarity. However, an elongating Pol II with a long RNA transcript becomes an even more potent and persistent roadblock while retaining the polarity, and the formation of an RNA-DNA hybrid mediates this enhancement. Surprisingly, we discovered that when a Pol II collides with the DNA fork head-on and becomes backtracked, an RNA-DNA hybrid can form on the lagging strand in front of Pol II, creating a topological lock that traps Pol II at the fork. TFIIS facilitates RNA-DNA hybrid removal by severing the connection of Pol II with the hybrid. We further demonstrate that this RNA-DNA hybrid can prime lagging strand replication by T7 DNA polymerase while Pol II is still bound to DNA. Our findings capture basal properties of the interactions of Pol II with a DNA fork, revealing significant implications for transcription-replication conflicts.
DNA复制和转录在同一DNA模板上同时发生，导致复制体与RNA聚合酶之间不可避免地产生冲突。这些冲突会使复制叉停滞，并威胁基因组稳定性。尽管众多研究表明，与同向冲突相比，迎头冲突更具危害性，也更易于促进R环形成，但RNA聚合酶阻碍极性的根本原因仍不清楚，而且这些R环的结构也只是推测性的。在这项研究中，我们使用一个简单的模型系统来解决这个复杂问题，通过检测Pol II对经由机械解链推进的DNA叉的阻碍，以此模拟复制体的前进过程。我们发现，即使转录本尺寸最小，Pol II在迎头构型中也能更稳定地结合以抵抗去除，这表明Pol II阻碍具有内在极性。然而，带有长RNA转录本的延伸中的Pol II会成为一个更强效且持久的阻碍，同时保留极性，并且RNA-DNA杂交体的形成介导了这种增强作用。令人惊讶的是，我们发现当Pol II与DNA叉迎头碰撞并发生回溯时，一个RNA-DNA杂交体可以在Pol II前方的后随链上形成，产生一个拓扑锁定，将Pol II困在叉处。TFIIS通过切断Pol II与杂交体的连接来促进RNA-DNA杂交体的去除。我们进一步证明，在Pol II仍与DNA结合时，这种RNA-DNA杂交体可以通过T7 DNA聚合酶引发后随链复制。我们的研究结果揭示了Pol II与DNA叉相互作用的基本特性，对转录-复制冲突具有重要意义。
