Regulation of Supercoil Unwinding by Topoisomerase 1B

描述（由申请人提供）：维持DNA超卷曲的稳态水平对多种生物过程至关重要。由于转录或复制叉的移动而产生的超级线圈的形成速度和由于沿DNA扩散的正负超级线圈波的碰撞而导致的超级线圈消失的速度之间存在着不断的竞争。1B型DNA拓扑异构酶（Topo 1B）的功能是去除正、负超级线圈。Topo1B与单链双链DNA形成自由可逆的共价磷酸酪氨酸连锁，导致DNA主干断裂，允许超卷曲DNA通过与下游旋转DNA部分的瞬时酶相互作用以受控的方式展开。人类拓扑- dna共价复合物是广泛使用的抗癌药物伊立替康和拓扑替康的唯一靶点，它们破坏超级线圈的解绕。尽管Topo-DNA复合物在调节超螺旋密度和抗癌药理学中发挥着核心作用，但人们对这种酶如何感知超螺旋密度以维持最佳的超螺旋稳态水平知之甚少，更重要的是，小分子如何干扰这一过程。本研究旨在阐明Topo1B活性如何响应DNA超卷曲的离散变化，并确定干扰拓扑异构酶催化的超卷曲的小分子的作用机制。具体目标是使用含有单个Topo1B识别位点的独特DNA小圆底物和可变数量的超线圈，研究超线圈对结合亲和力的影响（通过荧光各向异性），以及超线圈解绕的动力学（通过基于凝胶的离散超线圈中间体观察）。从超级线圈解绕的动力学模拟数据中，计算出DNA分裂、解绕和还原的速率常数。这些研究还将扩展到探索酶与下游DNA主链旋转部分之间静电相互作用的作用，使用甲基膦酸盐取代小环DNA和与该DNA区域相互作用的带正电的蛋白质残基的突变。为了模拟DNA旋转过程中结合的细胞蛋白的影响，本研究将通过将分子量增加的蛋白质附着在旋转DNA的特定结合位点上，来测量DNA旋转阻力对超级线圈解绕的影响。这些相同的实验将扩展到研究小分子抑制剂的机制及其对超级线圈展开效率的影响。

DESCRIPTION (provided by applicant): Maintenance of a steady-state level of DNA supercoiling is essential for diverse biological processes. There is constant competition between the rate of supercoil formation arising from moving transcription or replication forks, and the rat of supercoil disappearance due to collision of negative and positive supercoil waves diffusing along the DNA. Removal of both positive and negative supercoils is the function of type 1B DNA topoisomerase enzymes (Topo 1B). Topo1B forms a freely reversible covalent phosphotyrosine linkage with a single strand of duplex DNA, resulting in a break in the DNA backbone that allows supercoiled DNA to unwind in a controlled fashion using transient enzyme interactions with the downstream rotating DNA portion. Human Topo-DNA covalent complex forms the sole target for the widely used anticancer drugs irinotecan and topotecan, which disrupt supercoil unwinding. Despite the central role of the Topo-DNA complex in regulating superhelical density and in anticancer pharmacology, little is known about how the enzyme senses superhelical density to maintain an optimal steady-state level of supercoiling, and importantly, how small molecules interfere with this process. This proposal aims to elucidate how Topo1B activity responds to discrete changes in DNA supercoiling and to determine the mechanism of action of small molecules that perturb topoisomerase-catalyzed supercoil unwinding. The specific aims are to use a unique DNA minicircle substrate containing a single Topo1B recognition site, and a variable number of supercoils, to study the effect of supercoiling on binding affinity (via fluorescence anisotropy), and the kinetics of supercoil unwinding (via gel-based observation of discrete supercoiled intermediates). From kinetic simulations of the supercoil unwinding data, rate constants for DNA cleavage, unwinding, and religation will be calculated. These studies will also be extended to explore the role of electrostatic interactions between the enzyme and the rotating portion of the downstream DNA backbone using both methylphosphonate substitutions of the minicircle DNA and mutations of positively charged protein residues that interact with this DNA region. To simulate the effects of bound cellular proteins during DNA rotation, this study will measure the effect of DNA rotational drag on supercoil unwinding by affixing proteins with increasing molecular weight to a specific binding site on the rotating DNA. These same experiments will be expanded to study the mechanism of small molecule inhibitors and their effect on the efficiency of supercoil unwinding.