Sattin Bernie D, Goh M Cynthia
Department of Chemistry, University of Toronto, Toronto, Ontario, Canada.
Biophys J. 2004 Nov;87(5):3430-6. doi: 10.1529/biophysj.104.045567. Epub 2004 Aug 17.
The formation of the RecA/DNA nucleofilament on nicked circular double stranded (ds) DNA in the presence of ATPgammaS was studied using the atomic force microscope (AFM) at nanometer resolution. The AFM allowed simultaneous observation of both dsDNA substrate and RecA protein-coated sections such that they are highly distinguishable. Using a time series of images, the complex formation was monitored. AFM imaging provided direct evidence that assembly of the nucleofilaments occurs via a nucleation and growth mechanism. The nucleation step is much slower than the growth phase, as demonstrated by the predominance of naked dsDNA at early and middle time points, followed by the rapid appearance of partially then fully formed complexes. Observation of the formation of nucleation sites without accompanying growth on unnicked dsDNA enabled an estimate of the nucleation rate, of 5 x 10(-5) RecA min(-1) bp(-1). The published model for the analysis of RecA assembly on dsDNA deduces a single kinetic parameter that prevents the separate determination of nucleation rate and growth rate. By directly measuring the nucleation rate with the AFM, this model is employed to determine a growth rate of 202 min(-1). These AFM results provide the first direct evidence of previous results on complex formation obtained only by indirect means.
利用原子力显微镜（AFM）在纳米分辨率下研究了在ATPγS存在的情况下，切口环状双链（ds）DNA上RecA/DNA核丝的形成。AFM能够同时观察dsDNA底物和RecA蛋白包被的部分，使它们具有高度可区分性。通过一系列时间图像，监测复合物的形成。AFM成像提供了直接证据，证明核丝的组装是通过成核和生长机制发生的。成核步骤比生长阶段慢得多，这在早期和中期时间点裸dsDNA占主导地位得到证明，随后部分然后完全形成的复合物迅速出现。在未切口的dsDNA上观察到成核位点的形成而没有伴随生长，从而能够估计成核速率为5×10^(-5) RecA min^(-1) bp^(-1)。已发表的用于分析dsDNA上RecA组装的模型推导出一个单一动力学参数，这使得无法分别确定成核速率和生长速率。通过用AFM直接测量成核速率，该模型被用于确定生长速率为202 min^(-1)。这些AFM结果为以前仅通过间接方法获得的关于复合物形成的结果提供了首个直接证据。
