Regulation of Supercoil Unwinding by Topoisomerase 1B

描述（由申请人提供）：维持DNA超螺旋的稳态水平对于不同的生物过程至关重要。在由移动的转录或复制叉引起的超螺旋形成的速率与由于沿着DNA扩散的负超螺旋波和正超螺旋波的碰撞而引起的超螺旋消失的速率之间存在恒定的竞争。去除正超螺旋和负超螺旋是1B型DNA拓扑异构酶（Topo 1B）的功能。Topo 1B与双链DNA的单链形成自由可逆的共价磷酸酪氨酸连接，导致DNA骨架断裂，从而允许超螺旋DNA使用与下游旋转DNA部分的瞬时酶相互作用以受控方式解旋。人Topo-DNA共价复合物形成广泛使用的抗癌药物伊立替康和拓扑替康的唯一靶标，其破坏超螺旋解旋。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重要作用，但人们对酶如何感知超螺旋密度以维持超螺旋的最佳稳态水平知之甚少，重要的是，小分子如何干扰这一过程。该提案旨在阐明Topo 1B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螺旋解旋的小分子的作用机制。具体的目的是使用一个独特的DNA微环基板含有一个单一的Topo 1B识别位点，和一个变量的超螺旋，研究超螺旋结合亲和力的影响（通过荧光各向异性），和超螺旋解旋的动力学（通过基于凝胶的观察离散的超螺旋中间体）。根据超螺旋解旋数据的动力学模拟，将计算DNA切割、解旋和重新连接的速率常数。这些研究还将扩展到探索酶和旋转部分的下游DNA骨架使用甲基膦酸酯取代的小环DNA和带正电荷的蛋白质残基的突变，与此DNA区域相互作用之间的静电相互作用的作用。为了模拟DNA旋转过程中结合的细胞蛋白质的影响，本研究将通过将分子量增加的蛋白质附着到旋转DNA上的特定结合位点来测量DNA旋转阻力对超螺旋解旋的影响。这些相同的实验将扩大到研究小分子抑制剂的机制及其对超螺旋解旋效率的影响。

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.