Investigating the interplay between SMC complexes and Topoisomerase II

DNA是细胞中所有遗传信息的储存库。为了编码所有这些信息，DNA分子非常长。例如，一个人类细胞在直径小于20微米的细胞核中含有近200万个DNA。不仅所有的DNA都必须被打包到细胞核中，而且它的组织必须不断地重组，这样它的包装才能在细胞周期的大部分时间里促进正常的基因表达，然后在有丝分裂期间被紧密包装，以一种允许所有染色体忠实地分离到子细胞的方式。在细胞周期的不同阶段，染色体组织不当会导致染色体脆弱、染色体数目异常和细胞死亡。通常与癌症、衰老和人类生长障碍有关的特征。两种类型的蛋白质复合物似乎是建立和维持染色体组织所必需的，SMC复合物和II型拓扑异构酶（Top2）。古老的SMC复合体家族存在于所有生命王国中，在那里它们需要适当地构建遗传遗传的染色体。真核SMC复合体内聚蛋白和凝聚蛋白都被认为是通过促进DNA沿染色体形成环来组织染色体的。II型拓扑异构酶也存在于所有类型的细胞中，它们被认为通过允许DNA的一部分穿过另一部分来组织DNA，促进染色体的解开，并减轻DNA上积累的任何DNA拓扑压力。真核生物II型拓扑异构酶Top2被认为在染色体结构中具有酶和结构作用。然而，迄今为止对SMC复合体和Top2如何共同作用以动态组织染色体结构的分析产生了看似矛盾的结果。其中一些研究表明，SMC复合物和Top2相互作用，促进适当的基因表达和染色体分离。然而，其他人发现它们也可以独立甚至拮抗地维持正常的染色体结构。这些对比结果表明，在Top2和不同的SMC复合物之间存在复杂的、潜在的环境依赖的相互作用。了解这种相互作用的本质对于这两个古老的DNA操纵机器如何协同工作来组织DNA并确保正常的生物功能至关重要。事实上，编码Top2和SMC蛋白的基因突变与癌症的进展和动植物的生长密切相关。在本研究中，我们将研究SMC复合物和Top2在染色体结构上的相互作用和相互依赖。最近，我们利用Hi-C染色体构象技术表明，黏结蛋白和凝聚蛋白以不同的方式组织出芽酵母染色体的结构。在这里，我们将研究Top2是如何改变染色体结构的，以及这种变化是如何由不同的SMC复合物内聚蛋白和凝聚蛋白调节的。我们将继续研究染色体结构的Top2依赖性变化是否需要Top2的酶活性或仅与染色体结合，以及这些是否由SMC复合物调节。最后，我们将研究DNA超卷曲及其由Top2和潜在的SMCs调控是否组织染色体。总之，本提案旨在提供一个全面和连贯的分析，以了解Top2和SMC复合物内聚蛋白和凝聚蛋白如何相互作用以产生功能性染色体结构的基本DNA结构活性。

DNA is the repository for all the genetic information of a cell. To encode all this information DNA molecules are extraordinarily long. For example, a human cell contains nearly 2m of DNA in a nucleus smaller than 20 microns in diameter. Not only must all this DNA be packed into the nucleus but its organisation must be constantly re-organised so that its packaging can promote normal gene expression during most of the cell cycle before being tightly packaged during mitosis in a manner that allows faithful segregation of all chromosomes to daughter cells. Failure to appropriately organise chromosomes during the different stages of the cell cycle leads to chromosome fragility, aberrant chromosome numbers and cell death. Features often associated with cancer, ageing and human growth disorders.Two types of protein complexes appear to be essential to establish and maintain chromosomal organisation, SMC complexes and type II topoisomerases (Top2). The ancient family of SMC complexes are found in all the kingdoms of life, where they are required to appropriately structure chromosomes for genetic inheritance. Both the eukaryotic SMC complexes cohesin and condensin are thought to organise chromosomes by promoting DNA looping along chromosomes. Type II topoisomerases are also found in all cell types where they are thought to organize DNA by allowing one section of DNA to pass through another, promoting untangling of chromosomes and relaxing any DNA topological stress that builds up on the DNA. The eukaryotic type II topoisomerase Top2 is proposed to have both an enzymatic and structural role in chromosome structure. However, analysis to date of how SMC complexes and Top2 may work together to dynamically organise chromosome structure has produced seemingly contradictory results. Some of these studies indicate that SMC complexes and Top2 work in concert with each other to promote proper gene expression and chromosome segregation. However, others have found that they can also operate independently or even antagonistically in the maintenance of normal chromosome structure. These contrasting results indicate a complex and potentially context dependent interplay between Top2 and the different SMC complexes. Understanding the nature of this interplay is crucial for how these two ancient DNA manipulating machines work together to organise DNA and ensure normal biological function. Indeed, mutations in the genes encoding Top2 and SMC proteins are closely linked with cancer progression and the growth of animals and plants. In this proposal, we will investigate the interplay and co-dependencies of SMC complexes and Top2 on chromosome structure. Recently we have used the Hi-C chromosome conformation technique to show that cohesin and condensin organise the structure of budding yeast chromosomes in distinct ways. Here, we will examine how chromosome structure is altered by Top2 and how such changes are regulated by the different SMC complexes cohesin and condensin. We will go onto to examine if Top2 dependent changes to chromosome structure require the enzymatic activity of Top2 or only its binding to chromosomes and if these are regulated by SMC complexes. Finally, we will examine if DNA supercoiling and its regulation by Top2, and potentially SMCs, organizes chromosomes.Together this proposal aims to provide a comprehensive and coherent analysis of how the fundamental DNA structuring activities of Top2 and the SMC complexes cohesin and condensin interact to generate functional chromosome structure.