Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers
Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers
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DOI：
10.1038/89646
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发表时间：
2001-07-01
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影响因子：
--
通讯作者：
Greve, J
中科院分区：
文献类型：
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作者：
Bennink, ML;Leuba, SH;Greve, J
单染色质纤维直接组装在光镊装置的流动池中。一个单一的h噬菌体DNA分子，悬浮在两个聚苯乙烯珠之间，暴露于非洲爪蟾卵提取物，导致染色质组装伴随明显缩短的DNA分子。组装是力依赖性的，并且在超过10 pN的力下不能发生。组装的单个染色质纤维通过其中一个珠的受控运动进行拉伸，其中分子中产生的力被连续监测，第二个珠被捕获在光学陷阱中。在纤维拉伸时，力显示出离散的突然下降，反映了纤维结构中的离散开放事件。这些开放事件在纤维长度的增量类似于65 mn的情况下被量化，并且归因于DNA从单个组蛋白八聚体周围的解缠绕。在不存在蛋提取物的情况下纤维的重复拉伸和松弛表明组蛋白八聚体的损失是不可逆的。对于单个核小体破坏测量的力在20-40 pN的范围内，与对于RNA和DNA聚合酶报道的力相当。
Single chromatin fibers were assembled directly in the flow cell of an optical tweezers setup. A single h phage DNA molecule, suspended between two polystyrene beads, was exposed to a Xenopus laevis egg extract, leading to chromatin assembly with concomitant apparent shortening of the DNA molecule. Assembly was force-dependent and could not take place at forces exceeding 10 pN, The assembled single chromatin fiber was subjected to stretching by controlled movement of one of the beads with the force generated in the molecule continuously monitored with the second bead trapped in the optical trap. The force displayed discrete, sudden drops upon fiber stretching, reflecting discrete opening events in fiber structure. These opening events were quantized at increments in fiber length of similar to 65 mn and are attributed to unwrapping of the DNA from around individual histone octamers, Repeated stretching and relaxing of the fiber in the absence of egg extract showed that the loss of histone octamers was irreversible. The forces measured for Individual nucleosome disruptions are in the range of 20-40 pN, comparable to forces reported for RNA- and DNA-polymerases.