Senavirathne Gayan, Mahto Santosh K, Hanne Jeungphill, O'Brian Daniel, Fishel Richard
Department of Cancer Biology and Genetics, The Ohio State University Medical Center, Columbus, OH 43210, USA.
Department of Cancer Biology and Genetics, The Ohio State University Medical Center, Columbus, OH 43210, USA
Nucleic Acids Res. 2017 Jan 25;45(2):685-698. doi: 10.1093/nar/gkw920. Epub 2016 Oct 13.
Wrapping of genomic DNA into nucleosomes poses thermodynamic and kinetic barriers to biological processes such as replication, transcription, repair and recombination. Previous biochemical studies have demonstrated that in the presence of adenosine triphosphate (ATP) the human RAD51 (HsRAD51) recombinase can form a nucleoprotein filament (NPF) on double-stranded DNA (dsDNA) that is capable of unwrapping the nucleosomal DNA from the histone octamer (HO). Here, we have used single molecule Förster Resonance Energy Transfer (smFRET) to examine the real time nucleosome dynamics in the presence of the HsRAD51 NPF. We show that oligomerization of HsRAD51 leads to stepwise, but stochastic unwrapping of the DNA from the HO in the presence of ATP. The highly reversible dynamics observed in single-molecule trajectories suggests an antagonistic mechanism between HsRAD51 binding and rewrapping of the DNA around the HO. These stochastic dynamics were independent of the nucleosomal DNA sequence or the asymmetry created by the presence of a linker DNA. We also observed sliding and rotational oscillations of the HO with respect to the nucleosomal DNA. These studies underline the dynamic nature of even tightly associated protein-DNA complexes such as nucleosomes.
将基因组DNA包裹到核小体中对诸如复制、转录、修复和重组等生物过程构成了热力学和动力学障碍。先前的生化研究表明，在三磷酸腺苷（ATP）存在的情况下，人类RAD51（HsRAD51）重组酶可在双链DNA（dsDNA）上形成核蛋白丝（NPF），该核蛋白丝能够从组蛋白八聚体（HO）上解开核小体DNA。在此，我们利用单分子荧光共振能量转移（smFRET）来检测存在HsRAD51 NPF时的实时核小体动力学。我们发现，在ATP存在的情况下，HsRAD51的寡聚化导致DNA从HO上逐步但随机地解开。在单分子轨迹中观察到的高度可逆动力学表明，HsRAD51结合与DNA围绕HO重新包裹之间存在拮抗机制。这些随机动力学与核小体DNA序列或连接DNA的存在所产生的不对称性无关。我们还观察到HO相对于核小体DNA的滑动和旋转振荡。这些研究强调了即使是像核小体这样紧密结合的蛋白质 - DNA复合物的动态性质。
