Single-molecule mapping of replisome progression.
Single-molecule mapping of replisome progression.
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DOI：
10.1016/j.molcel.2022.02.010
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发表时间：
2022-04-07
期刊：
影响因子：
16
通讯作者：
Whitehouse I
中科院分区：
文献类型：
--
作者：
Claussin C;Vazquez J;Whitehouse I
DNA复制的基本方面，如复制停滞位点的解剖，复制体如何受到基因转录的影响，以及姐妹复制体的进展是否协调，都知之甚少。现有的技术不允许在染色质上精确定位单个复制体的位置。我们已经开发了一种称为复制子测序的方法，该方法需要通过复制叉处的受控核酸酶切割来切除全长复制子。使用Nanopore对复制子进行测序，Nanopore提供长DNA的单分子读数。使用复制子测序，我们发现姐妹复制体自主发挥功能，但通过染色质的进展具有显着的一致性。遇到障碍的复制分叉会暂停一小段时间，但很快就会恢复合成。解旋酶Rrm3在减轻蛋白质屏障的影响和促进有效终止方面都起着关键作用。复制子测序提供了一种高分辨率的方法来定义单个复制体如何在基因组中移动。Claussin等人开发了一种方法，能够以单分子和单核苷酸的精度精确地绘制DNA合成的位置。“复制子测序”提供了一个新的工具来研究DNA复制的许多方面，包括：起始，延伸，终止和复制障碍的各种影响。
Fundamental aspects of DNA replication, such as the anatomy of replication stall sites, how replisomes are influenced by gene transcription and whether the progression of sister replisomes is coordinated are poorly understood. Available techniques do not allow the precise mapping of the positions of individual replisomes on chromatin. We have developed a method called Replicon-seq that entails the excision of full-length replicons by controlled nuclease cleavage at replication forks. Replicons are sequenced using Nanopore, which provides a single-molecule readout of long DNA. Using Replicon-seq, we found that sister replisomes function autonomously yet progress through chromatin with remarkable consistency. Replication forks that encounter obstacles pause for a short duration but rapidly resume synthesis. The helicase Rrm3 plays a critical role both in mitigating the effect of protein barriers and facilitating efficient termination. Replicon-seq provides a high-resolution means of defining how individual replisomes move across the genome. Claussin et al have developed a method that is able to precisely map the locations of DNA synthesis with single-molecule and single-nucleotide precision. “Replicon-seq” provides a new tool to study many aspects of DNA replication, including: initiation, elongation, termination and the various effects of replication impediments.
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