Protein-DNA interactions in high speed AFM: single molecule diffusion analysis of human RAD54.
Protein-DNA interactions in high speed AFM: single molecule diffusion analysis of human RAD54.
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DOI：
10.1039/c1ib00039j
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发表时间：
2011-10
期刊：
影响因子：
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通讯作者：
H. Sánchez;Yuki Suzuki;M. Yokokawa;K. Takeyasu;C. Wyman
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文献类型：
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作者：
H. Sánchez;Yuki Suzuki;M. Yokokawa;K. Takeyasu;C. Wyman
高速AFM（原子力显微镜，也称为扫描力显微镜）提供单个分子的纳米空间分辨率和亚秒时间分辨率图像。我们利用这些功能来研究扩散和运动活性的RAD 54 DNA修复因子。人RAD 54在重组DNA修复的关键步骤中发挥作用。它是Swi 2/Snf 2染色质重塑家族的成员，其使用ATP水解在DNA上易位。DNA-蛋白质相互作用的详细单分子描述显示了中间状态和可变状态的分布，通常被系综平均所隐藏。我们使用单粒子跟踪测量单个蛋白质的运动，并观察到随机游走受到成像缓冲液成分的影响。非布朗扩散事件的特点是在存在和不存在的核苷酸辅因子。固定在表面上的双链DNA起到了减少布朗运动的陷阱的作用。通过高速AFM可视化DNA上的明显短程载玻片和啤酒花。这些短程相互作用通常无法通过基于光学分辨率的其他方法获得。RAD 54单体表现出与运动活动无关的扩散行为。
High-speed AFM (atomic force microscopy also called scanning force microscopy) provides nanometre spatial resolution and sub-second temporal resolution images of individual molecules. We exploit these features to study diffusion and motor activity of the RAD54 DNA repair factor. Human RAD54 functions at critical steps in recombinational-DNA repair. It is a member of the Swi2/Snf2 family of chromatin remodelers that translocate on DNA using ATP hydrolysis. A detailed single molecular description of DNA-protein interactions shows intermediate states and distribution of variable states, usually hidden by ensemble averaging. We measured the motion of individual proteins using single-particle tracking and observed that random walks were affected by imaging-buffer composition. Non-Brownian diffusion events were characterized in the presence and in the absence of nucleotide cofactors. Double-stranded DNA immobilized on the surface functioned as a trap reducing Brownian motion. Distinct short range slides and hops on DNA were visualized by high-speed AFM. These short-range interactions were usually inaccessible by other methods based on optical resolution. RAD54 monomers displayed a diffusive behavior unrelated to the motor activity.