How does Condensin mediate topological change during mitosis?

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Maintaining genome stability is essential for normal cellular function. Cellular defects that lead to loss or duplication of genetic information can lead to cell death, cancer or premature aging. Maintenance of genome stability is a particularly acute problem in dividing cells. Every time a cell divides, every one of the chromosomes that makes up the genome must be perfectly copied. The two copies must then fully resolved from one other and finally one copy of every chromosome transported into each daughter cell. Failure to faithfully complete any or these steps could have disastrous effects on genome stability.Understanding how cellular proteins work together to maintain genome instability is therefore crucial to understanding why cells become cancerous or defective with age. The SMC family of proteins appear crucial to genome stability in all organisms from bacteria to humans. The SMC proteins form several different complexes in the cell but in all cases they appear to function by influencing chromosome structure.One of the SMC complexes called condensin appears to be important for chromosomal compaction in mitosis. How condensin compacts chromosomes remains enigmatic although it has been shown in the test tube to generate supercoiled "spring like" structures in DNA. Recently I have shown that similar structures are formed in cells just before the chromosomes are segregated. This change in chromosome structure promotes the resolution of the chromosomes and so appears crucial for chromosome stability.The work in this proposal aims to demonstrate how the condensin complex changes chromosome structure in order to promote genome stability. It will first use molecular biology techniques to modify distinct regions of the condensin factors SMC2 and SMC4. We will then use these modified proteins to assess how specific aspects of SMC2 and SMC4 function are required to generate supercoiled structures in the cells. The ability of the altered proteins to generate supercoiled structures will then be related to how condensin alters chromosome organisation and the cells ability to ensure that one whole copy of each chromosome is segregated to each daughter cell. These experiments aim to provide a detailed description of what aspects of SMC/condensin function are required to change chromosome structure inside cells. We will then use this information to recreate the situation in a test tube and so generate a system where we will be able to fully describe how condensin achieves its function.Using this combination of approaches this work aims to provide unique insights into how SMC/condensin proteins, and potentially all other SMC proteins help maintain genome stability. The information generated by this project will be of general interest not only to researchers studying genome stability but also for research into ageing and cancer evolution.