Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Cell Rep. 2019 Apr 9;27(2):374-386.e4. doi: 10.1016/j.celrep.2019.03.035.
The SWR1C chromatin remodeling enzyme catalyzes ATP-dependent replacement of nucleosomal H2A with the H2A.Z variant, regulating key DNA-mediated processes such as transcription and DNA repair. Here, we investigate the transient kinetic mechanism of the histone exchange reaction, employing ensemble FRET, fluorescence correlation spectroscopy (FCS), and the steady-state kinetics of ATP hydrolysis. Our studies indicate that SWR1C modulates nucleosome dynamics on both the millisecond and microsecond timescales, poising the nucleosome for the dimer exchange reaction. The transient kinetic analysis of the remodeling reaction performed under single turnover conditions unraveled a striking asymmetry in the ATP-dependent replacement of nucleosomal dimers, promoted by localized DNA unwrapping. Taken together, our transient kinetic studies identify intermediates and provide crucial insights into the SWR1C-catalyzed dimer exchange reaction and shed light on how the mechanics of H2A.Z deposition might contribute to transcriptional regulation in vivo.
SWR1C 染色质重塑酶催化 ATP 依赖性核小体 H2A 被 H2A.Z 变体取代，调节关键的 DNA 介导过程，如转录和 DNA 修复。在这里，我们通过荧光各向异性（FRET）、荧光相关光谱（FCS）和 ATP 水解的稳态动力学研究了组蛋白交换反应的瞬态动力学机制。我们的研究表明，SWR1C 在毫秒和微秒时间尺度上调节核小体动力学，使核小体为二聚体交换反应做好准备。在单轮条件下进行的重塑反应的瞬态动力学分析揭示了局部 DNA 解缠对核小体二聚体 ATP 依赖性取代的惊人不对称性。总之，我们的瞬态动力学研究确定了中间体，并为 SWR1C 催化的二聚体交换反应提供了关键见解，并阐明了 H2A.Z 沉积的力学如何有助于体内转录调控。
