Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament.
Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament.
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DOI：
10.1093/nar/gkt425
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发表时间：
2013-08
影响因子：
14.9
通讯作者：
Dekker NH
中科院分区：
文献类型：
--
作者：
Lee M;Lipfert J;Sanchez H;Wyman C;Dekker NH
人类RAD51是DNA同源重组修复过程中的关键蛋白。它组装到DNA上，引起DNA结构的变化，导致核蛋白细丝的形成，形成链交换的基础。在这里，我们确定了RAD51-dsDNA细丝的结构和机械性能。我们的测量使用了两种最新开发的磁性镊子分析方法，自由轨道磁性镊子和磁性扭矩镊子，旨在测量单个分子的扭曲和扭矩。通过直接监测RAD51结合上DNA扭曲的变化，我们确定每个RAD51单体的解旋角度为45°，与其细菌同源物RecA的解旋角度定量一致。对RAD51-dsDNA细丝扭曲时所产生的扭矩的测量表明，在抑制ATP水解的条件下，RAD51-dsDNA细丝的扭转持续长度超过其RecA对应细丝的三倍。对不同二价离子和核苷酸辅因子组合的纤维扭转硬度的研究表明，钙离子除了抑制ATPase活性外，还在增加纤维扭转硬度方面起着关键作用。这些对RAD51造成的DNA扭曲和累积的机械应力的定量测量表明，RAD51核蛋白细丝内的化学和机械相互作用之间存在微调的相互作用。
Human RAD51 is a key protein in the repair of DNA by homologous recombination. Its assembly onto DNA, which induces changes in DNA structure, results in the formation of a nucleoprotein filament that forms the basis of strand exchange. Here, we determine the structural and mechanical properties of RAD51-dsDNA filaments. Our measurements use two recently developed magnetic tweezers assays, freely orbiting magnetic tweezers and magnetic torque tweezers, designed to measure the twist and torque of individual molecules. By directly monitoring changes in DNA twist on RAD51 binding, we determine the unwinding angle per RAD51 monomer to be 45°, in quantitative agreement with that of its bacterial homolog, RecA. Measurements of the torque that is built up when RAD51-dsDNA filaments are twisted show that under conditions that suppress ATP hydrolysis the torsional persistence length of the RAD51-dsDNA filament exceeds that of its RecA counterpart by a factor of three. Examination of the filament’s torsional stiffness for different combinations of divalent ions and nucleotide cofactors reveals that the Ca2+ ion, apart from suppressing ATPase activity, plays a key role in increasing the torsional stiffness of the filament. These quantitative measurements of RAD51-imposed DNA distortions and accumulated mechanical stress suggest a finely tuned interplay between chemical and mechanical interactions within the RAD51 nucleoprotein filament.
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影响因子：
16.8
作者：
Conway, AB;Lynch, TW;Rice, PA
通讯作者：
Rice, PA
影响因子：
8.6
作者：
Forth, Scott;Deufel, Christopher;Wang, Michelle D.
通讯作者：
Wang, Michelle D.
影响因子：
14.9
作者：
Fornander, Louise H.;Frykholm, Karolin;Norden, Bengt
通讯作者：
Norden, Bengt
DOI：
10.1007/978-1-60761-340-4_7
发表时间：
2009-01-01
期刊：
DNA TOPOISOMERASES: METHODS AND PROTOCOLS
影响因子：
--
作者：
Lipfert, Jan;Koster, Daniel A.;Dekker, Nynke H.
通讯作者：
Dekker, Nynke H.
影响因子：
1.6
作者：
Lipfert, Jan;Kerssemakers, Jacob J. W.;Dekker, Nynke H.
通讯作者：
Dekker, Nynke H.