Biton Yoav Y, Kumar Sandip, Dunlap David, Swigon David
Department of Mathematics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
PLoS One. 2014 May 6;9(5):e92475. doi: 10.1371/journal.pone.0092475. eCollection 2014.
Tethered particle motion (TPM) experiments can be used to detect time-resolved loop formation in a single DNA molecule by measuring changes in the length of a DNA tether. Interpretation of such experiments is greatly aided by computer simulations of DNA looping which allow one to analyze the structure of the looped DNA and estimate DNA-protein binding constants specific for the loop formation process. We here present a new Monte Carlo scheme for accurate simulation of DNA configurations subject to geometric constraints and apply this method to Lac repressor mediated DNA looping, comparing the simulation results with new experimental data obtained by the TPM technique. Our simulations, taking into account the details of attachment of DNA ends and fluctuations of the looped subsegment of the DNA, reveal the origin of the double-peaked distribution of RMS values observed by TPM experiments by showing that the average RMS value for anti-parallel loop types is smaller than that of parallel loop types. The simulations also reveal that the looping probabilities for the anti-parallel loop types are significantly higher than those of the parallel loop types, even for loops of length 600 and 900 base pairs, and that the correct proportion between the heights of the peaks in the distribution can only be attained when loops with flexible Lac repressor conformation are taken into account. Comparison of the in silico and in vitro results yields estimates for the dissociation constants characterizing the binding affinity between O1 and Oid DNA operators and the dimeric arms of the Lac repressor.
系留粒子运动（TPM）实验可通过测量DNA系链长度的变化来检测单个DNA分子中时间分辨的环形成。DNA环化的计算机模拟极大地有助于此类实验的解释，它能让人们分析环化DNA的结构，并估计环形成过程中特定的DNA-蛋白质结合常数。我们在此提出一种新的蒙特卡罗方案，用于精确模拟受几何约束的DNA构型，并将此方法应用于乳糖阻遏物介导的DNA环化，将模拟结果与通过TPM技术获得的新实验数据进行比较。我们的模拟考虑了DNA末端的附着细节和DNA环化子片段的波动，通过表明反平行环类型的平均均方根（RMS）值小于平行环类型，揭示了TPM实验中观察到的RMS值双峰分布的起源。模拟还表明，即使对于长度为600和900个碱基对的环，反平行环类型的环化概率也显著高于平行环类型，并且只有在考虑具有灵活乳糖阻遏物构象的环时，才能获得分布中峰高之间的正确比例。计算机模拟结果与体外实验结果的比较得出了表征O1和Oid DNA操纵子与乳糖阻遏物二聚体臂之间结合亲和力的解离常数估计值。
