Regulation of Supercoil Unwinding by Topoisomerase 1B

拓扑异构酶 1B 对超螺旋解旋的调节

• 批准号: 8471547
• 负责人: Breeana Grogan Anderson
• 金额: $ 4.22万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-16 至 2015-05-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8471547
• 关键词: Acids; Active Sites; Address; Affect; Affinity; Antineoplastic Agents; Appearance; Binding; Binding Sites; Biological Assay; Biological Process; Charge; Chimeric Proteins; Clinical; Complex; DNA; DNA Topoisomerases; DNA biosynthesis; Data; Diffuse; Drug Targeting; Drug usage; Electrostatics; Enzyme Interaction; Enzymes; Event; Excision; Fluorescein; Fluorescence Anisotropy; Gel; Gene Mutation; Genetic Recombination; Genetic Transcription; Genome; Human; Individual; Kinetics; Label; Maintenance; Measurement; Measures; Molecular Weight; Mutate; Pharmaceutical Preparations; Pharmacology; Phenylalanine; Phosphotyrosine; Play; Positioning Attribute; Process; Property; Proteins; Rattus; Regulation; Role; Rotation; Simulate; Site; Smallpox Viruses; Structure; Superhelical DNA; Topoisomerase; Topotecan; Toxic effect; Tyrosine; Vertebral column; Zinc Fingers; antitumor agent; arginyllysine; base; controlled release; density; design; drug mechanism; enzyme activity; inhibitor/antagonist; innovation; irinotecan; methylphosphonate; mutant; novel; phosphonate; public health relevance; research study; simulation; small molecule

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.

描述（由申请人提供）：维持 DNA 超螺旋的稳态水平对于多种生物过程至关重要。由于移动转录或复制叉而产生的超螺旋形成速率与由于沿着DNA扩散的负超螺旋波和正超螺旋波的碰撞而导致的超螺旋消失之间存在着持续的竞争。 1B 型 DNA 拓扑异构酶 (Topo 1B) 的功能是去除正超螺旋和负超螺旋。 Topo1B 与单链双链 DNA 形成自由可逆的共价磷酸酪氨酸连接，导致 DNA 主链断裂，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标，它们会破坏超螺旋的解旋。尽管拓扑-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，但人们对该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，更重要的是，小分子如何干扰这一过程。该提案旨在阐明 Topo1B 活性如何响应 DNA 超螺旋的离散变化，并确定扰乱拓扑异构酶催化的超螺旋解旋的小分子的作用机制。具体目标是使用包含单个 Topo1B 识别位点和可变数量超螺旋的独特 DNA 微环底物，研究超螺旋对结合亲和力的影响（通过荧光各向异性），以及超螺旋解旋动力学（通过凝胶-基于离散超螺旋中间体的观察）。根据超螺旋解旋数据的动力学模拟，将计算 DNA 切割、解旋和再连接的速率常数。这些研究还将扩展到探索酶和下游 DNA 主链旋转部分之间静电相互作用的作用，使用小环 DNA 的甲基膦酸酯取代和与该 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固定到旋转 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。