Monitoring the compaction of single DNA molecules in Xenopus egg extract in real time.
Monitoring the compaction of single DNA molecules in Xenopus egg extract in real time.
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DOI：
10.1073/pnas.2221309120
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发表时间：
2023-03-21
影响因子：
11.1
通讯作者：
Heald, Rebecca
中科院分区：
文献类型：
--
作者：
Sun, Mingxuan;Amiri, Hossein;Tong, Alexander B.;Shintomi, Keishi;Hirano, Tatsuya;Bustamante, Carlos;Heald, Rebecca
染色体凝聚的机制知之甚少。我们结合爪蟾卵提取物与光镊检查压缩的单个DNA分子在生理条件下，支持细胞周期依赖的染色体组装在体外。核心组蛋白、连接组蛋白或凝聚蛋白的消耗具有不同的影响，这提供了对它们对凝聚染色质状态的个体贡献的深入了解。而凝聚素是驱动压实的主要参与者，组蛋白起稳定压实的DNA的作用。DNA压缩是有丝分裂过程中染色体的凝聚和分解所必需的，但对单个染色质因子对这一过程的相对贡献知之甚少。我们开发了一个生理的，无细胞系统，使用高速爪蟾卵提取物和光镊实时有丝分裂染色质纤维的形成和力诱导的拆卸单个DNA分子。与间期提取物相比，间期提取物使DNA压缩约60%，中期提取物使DNA长度减少90%以上，反映了这两种条件下全染色体形态的差异。耗尽的核心组蛋白伴侣ASF1，抑制核小体组装，中期纤维压实的最终程度降低了29%，而耗尽的连接组蛋白H1有更大的影响，减少总压缩40%。与对照组相比，两种消耗都降低了压实速率，导致更短的解压缩时间，并增加了力诱导纤维分解的速度。相反，中期提取物中凝聚素的耗尽强烈抑制了纤维组装，导致在高力下迅速逆转的瞬时压实事件。总之，这些研究结果支持了一个推测模型，其中凝聚蛋白在有丝分裂DNA压缩中起主要作用，而核心和接头组蛋白在环挤出过程中起减少滑动的作用，并调节DNA压缩的程度。
Mechanisms underlying chromosome condensation are poorly understood. We combined Xenopus egg extracts with optical tweezers to examine the compaction of single DNA molecules under physiological conditions that support cell cycle–dependent chromosome assembly in vitro. Depletion of core histones, linker histones, or condensins had distinct effects that provide insight into their individual contributions to the condensed chromatin state. Whereas condensin is the major player driving compaction, histones act to stabilize the compacted DNA. DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed Xenopus egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.
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DOI：
10.1126/science.aar7831
发表时间：
2018-04-06
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Ganji M;Shaltiel IA;Bisht S;Kim E;Kalichava A;Haering CH;Dekker C
通讯作者：
Dekker C
DNA超螺旋对染色质结构的影响。
影响因子：
--
作者：
Corless, Samuel;Gilbert, Nick
通讯作者：
Gilbert, Nick
影响因子：
3.7
作者：
Freedman, Benjamin S.;Miller, Kelly E.;Heald, Rebecca
通讯作者：
Heald, Rebecca
影响因子：
7.8
作者：
Walther, Nike;Hossain, M. Julius;Ellenberg, Jan
通讯作者：
Ellenberg, Jan
影响因子：
--
作者：
Rankin, Susannah
通讯作者：
Rankin, Susannah