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

IF 14.5 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Molecular Cell Pub Date : 2024-09-04 DOI:10.1016/j.molcel.2024.08.004
Rose Westhorpe, Johann J. Roske, Joseph T.P. Yeeles
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Abstract

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.

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控制拓扑异构酶 1 分裂复合物复制叉停滞和崩溃的机制
拓扑异构酶 1 分裂复合物(Top1-ccs)由 DNA 蛋白交联和单链 DNA 断裂组成,可对 DNA 复制机制(复制体)产生重大影响。因此,捕获 Top1-ccs 的抑制剂被广泛用于研究和临床环境中,以产生 DNA 复制压力,但复制体在与 Top1-cc 碰撞时如何反应仍不清楚。通过重构由纯化蛋白组装而成的芽殖酵母复制体与特定位点的 Top1-cc 之间的碰撞,我们发现了复制叉停滞和崩溃的内在机制。我们发现,停滞的复制叉出奇地稳定,而且其稳定性受到 Top1 交联的模板链、叉保护复合体蛋白 Tof1-Csm3(人类 TIMELESS-TIPIN)以及复制叉汇聚的影响。此外,停滞叉的新生链图谱和低温电子显微镜(cryo-EM)证实,复制体重塑是对 Top1-ccs 最初反应的关键因素。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。
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来源期刊
Molecular Cell
Molecular Cell 生物-生化与分子生物学
CiteScore
26.00
自引率
3.80%
发文量
389
审稿时长
1 months
期刊介绍: Molecular Cell is a companion to Cell, the leading journal of biology and the highest-impact journal in the world. Launched in December 1997 and published monthly. Molecular Cell is dedicated to publishing cutting-edge research in molecular biology, focusing on fundamental cellular processes. The journal encompasses a wide range of topics, including DNA replication, recombination, and repair; Chromatin biology and genome organization; Transcription; RNA processing and decay; Non-coding RNA function; Translation; Protein folding, modification, and quality control; Signal transduction pathways; Cell cycle and checkpoints; Cell death; Autophagy; Metabolism.
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