Structural and mechanistic analysis of type II DNA topoisomerases

DNA拓扑异构酶是在生命的所有领域中发生的必需酶，参与调节DNA拓扑结构以促进如DNA复制、染色体分离和转录等基本过程。这种功能是基于它们建立瞬时DNA断裂并催化另一个DNA片段在重新连接之前通过断裂的能力。这个过程是如何发生的还不清楚，但酶已成为抗菌和抗癌化疗的关键目标。拓扑异构酶靶向药物通过破坏DNA切割-再连接过程起作用。在这里，我们将使用生物化学和结构的方法来研究这个问题。一个关键问题是二价阳离子（通常是Mg 2+）在DNA断裂-团聚中的作用。已知许多DNA核酸酶利用Mg 2+来催化切割，但拓扑异构酶能够以协调的方式组合联合收割机切割和连接，以允许DNA通过而不留下永久性断裂;这究竟是如何发生的尚不清楚。最近的生物化学和结构证据表明，有两个Mg 2+结合位点的酶上的每一个DNA链。然而，人们对精确的机制、其调节及其动态知之甚少。值得注意的是，尚不清楚两种金属离子是否必须同时结合，或者一种金属是否可以在位点之间移动以调节方式催化裂解/再连接。最近的结构/生物化学的进展，现在我们有机会解决周围的问题，金属在裂解-再连接的作用。我们得出的原理将适用于以这种方式利用金属的许多酶，并将有助于抗菌和抗癌药物的开发。主要的方法将是结构生物学（X射线晶体学）和酶学。

DNA topoisomerases are essential enzymes that occur across all domains of life, involved in regulating DNA topology to facilitate essential processes like DNA replication, chromosome segregation and transcription. This function is based on their ability to establish transient DNA breaks and catalyse the passage of another DNA segment through the break before religation. How this process occurs is not well-understood, but the enzymes have become key targets for antibacterial and anti-cancer chemotherapy. Topoisomerase-targeted drugs work by disrupting the DNA cleavage-religation process. Here we will use biochemical and structural approaches to investigate this problem. A key issue will be the role of divalent cations (generally Mg2+) in DNA breakage-reunion. Many DNA nucleases are known to utilise Mg2+ to catalyse cleavage, but topoisomerases are able to combine cleavage and ligation in a coordinated manner to allow DNA passage without leaving a permanent break; exactly how this occurs is not clear. Recent biochemical and structural evidence suggests there are two Mg2+-binding sites per DNA strand on the enzyme. However, the precise mechanism, its regulation and its dynamics are poorly understood. Notably it is unclear whether two metal ions must bind at the same time or one metal can move between sites to catalyse cleavage/religation in a regulated manner. Recent structural/biochemical advances now present us with the opportunity to address questions surrounding the role of metals in cleavage-religation. The principles we derive will be applicable to many enzymes that utilise metals in this way, and will help with the development of antibacterial and anti-cancer drugs. The main approaches will be structural biology (X-ray crystallography) and enzymology.