Regulation of Supercoil Unwinding by Topoisomerase 1B

描述(由申请人提供)：维持稳定的DNA超螺旋水平对于不同的生物过程是必不可少的。由于转录或复制分叉的移动而产生的超级线圈的形成速度与由于沿着DNA扩散的正负超线圈波的碰撞而消失的超级线圈的速度之间存在着持续的竞争。正负超级线圈的去除是1B型DNA拓扑异构酶(TOPO 1B)的功能。TopO1B与单链双链DNA形成自由可逆的共价磷酸酪氨酸键，导致DNA骨架断裂，使超螺旋DNA能够通过瞬时酶与下游旋转的DNA部分相互作用以受控的方式解开。人类Topo-DNA共价复合体是广泛使用的抗癌药物伊立替康和拓扑替康的唯一靶点，这两种药物扰乱了超级线圈的解离。尽管Topo-DNA复合体在调节超螺旋密度和抗癌药理学中发挥着核心作用，但人们对该酶如何感知超螺旋密度以维持最佳稳态超螺旋水平知之甚少，更重要的是，小分子如何干扰这一过程。这一建议旨在阐明TopO1B活性如何响应DNA超螺旋的离散变化，并确定干扰拓扑异构酶催化的超级螺旋解卷的小分子的作用机制。其具体目的是使用一种独特的DNA微环底物，该底物包含单个TopO1B识别位点和可变数量的超级螺旋，以研究超级螺旋对结合亲和力的影响(通过荧光各向异性)，以及超级螺旋解卷的动力学(通过对离散的超级螺旋中间体进行凝胶观察)。根据超级线圈解卷数据的动力学模拟，将计算DNA裂解、解卷和宗教的速率常数。这些研究还将扩大到探索酶和下游DNA骨架旋转部分之间的静电相互作用的作用，使用微环DNA的甲基膦取代和与该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旋转过程中结合细胞蛋白质的影响，本研究将通过将分子量增加的蛋白质附着到旋转DNA上的特定结合位置来测量DNA旋转阻力对超级线圈解卷的影响。这些相同的实验将扩展到研究小分子缓蚀剂的机理及其对超级线圈解卷效率的影响。

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.