Mechanisms controlling replication fork stalling and collapse at topoisomerase 1 cleavage complexes,Molecular Cell

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Mechanisms controlling replication fork stalling and collapse at topoisomerase 1 cleavage complexes
Molecular Cell ( IF 14.5 ) Pub Date : 2024-09-04 , DOI: 10.1016/j.molcel.2024.08.004
Rose Westhorpe ₁ , Johann J Roske ₁ , Joseph T P Yeeles ₁

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Topoisomerase 1 cleavage complexes (Top1-ccs) comprise a DNA-protein crosslink and a single-stranded DNA break that can significantly impact the DNA replication machinery (replisome). Consequently, inhibitors that trap Top1-ccs are used extensively in research and clinical settings to generate DNA replication stress, yet how the replisome responds upon collision with a Top1-cc remains obscure. By reconstituting collisions between budding yeast replisomes, assembled from purified proteins, and site-specific Top1-ccs, we have uncovered mechanisms underlying replication fork stalling and collapse. We find that stalled replication forks are surprisingly stable and that their stability is influenced by the template strand that Top1 is crosslinked to, the fork protection complex proteins Tof1-Csm3 (human TIMELESS-TIPIN), and the convergence of replication forks. Moreover, nascent-strand mapping and cryoelectron microscopy (cryo-EM) of stalled forks establishes replisome remodeling as a key factor in the initial response to Top1-ccs. These findings have important implications for the use of Top1 inhibitors in research and in the clinic.

中文翻译：

控制拓扑异构酶 1 切割复合物处复制叉停滞和崩溃的机制

拓扑异构酶 1 切割复合物（Top1-ccs）包括 DNA-蛋白质交联和单链 DNA 断裂，可显著影响 DNA 复制机制（复制体）。因此，捕获 Top1-cc 的抑制剂在研究和临床环境中被广泛用于产生 DNA 复制应激，但复制体在与 Top1-cc 碰撞时的反应仍然不清楚。通过重构由纯化蛋白质组装的出芽酵母复制体与位点特异性 Top1-ccs 之间的碰撞，我们揭示了复制叉停滞和崩溃的潜在机制。我们发现停滞的复制叉非常稳定，并且它们的稳定性受 Top1 交联的模板链、叉保护复合物蛋白 Tof1-Csm3 （人类 TIMELESS-TIPIN）和复制叉的收敛的影响。此外，停滞叉子的新生链映射和冷冻电子显微镜（cryo-EM）将复制体重塑确定为对 Top1-ccs 初始反应的关键因素。这些发现对 Top1 抑制剂在研究和临床中的应用具有重要意义。

更新日期：2024-09-04

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