Investigating the interplay between SMC complexes and Topoisomerase II

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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.