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首页> 外文期刊>Journal of Molecular Biology >Kinetics of site-site interactions in supercoiled DNA with bent sequences.
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Kinetics of site-site interactions in supercoiled DNA with bent sequences.

机译：具有弯曲序列的超螺旋DNA中位点-位点相互作用的动力学。

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摘要

A curved DNA segment is known to adopt a preferred end loop localization in superhelical (sc) DNA and thus may organize the overall conformation of the molecule. Through this process it influences the probability of site juxtaposition. We addressed the effect of a curvature on site-site interactions quantitatively by measuring the kinetics of cross-linking of two biotinylated positions in scDNA by streptavidin. The DNA was biotinylated at either symmetric or asymmetric positions with respect to a curved insert via triplex-forming oligonucleotides (TFOs) modified with biotin. We used a quench-flow device to mix the DNA with the protein and scanning force microscopy to quantify the reaction products. As a measure of the interaction probability, rate constants of cross-linking and local concentrations j(M) of one biotinylated site in the vicinity of the other were determined and compared to Monte Carlo simulations for corresponding DNAs. In good agreement with the simulations, a j(M) value of 1.74 microM between two sites 500bp apart was measured for an scDNA without curvature. When a curvature was centered between the sites, the interaction probability increased about twofold over the DNA without curvature, significantly less than expected from the simulations. However, the relative differences of the interaction probabilities due to varied biotin positions with respect to the curvature agreed quantitatively with the theory.
机译：已知弯曲的DNA区段在超螺旋（sc）DNA中采用优选的末端环定位，因此可以组织分子的整体构象。通过此过程，它会影响站点并置的可能性。我们通过测量链霉亲和素在scDNA中两个生物素化位置的交联动力学，定量解决了曲率对位点-位点相互作用的影响。通过生物素修饰的三链体形成寡核苷酸（TFO），DNA在相对于弯曲插入物的对称或不对称位置被生物素化。我们使用淬灭流装置将DNA与蛋白质混合，并用扫描力显微镜对反应产物进行定量。作为相互作用概率的度量，确定了一个生物素化位点在另一个附近的交联速率常数和局部浓度j（M），并将其与相应DNA的Monte Carlo模拟进行了比较。与模拟良好吻合，对于没有曲率的scDNA，测量到相距500bp的两个位点之间的j（M）值为1.74 microM。当曲率集中在两个位点之间时，相互作用概率比没有曲率的DNA大约增加了两倍，大大低于模拟的预期。然而，由于生物素位置相对于曲率的变化而引起的相互作用概率的相对差异与该理论定量地吻合。

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