How does Condensin mediate topological change during mitosis?

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.

The stability of chromosome inheritance is of paramount importance to all living organisms. This requires that in every cell division one complete copy of the parental genome must be segregated into each of the two daughter cells. In eukaryotes the Condensin complex is thought to promote chromosome inheritance through ordered compaction of the genome in mitosis. The mechanism of how condensin achieves this remains enigmatic. From in vitro studies of condensin complexes it has been proposed that compaction is achieved through the generation of positive superhelical stress in the DNA [1]. This idea has recently been supported by the observation that condensin dependent positive supercoiling of DNA occurs in yeast cells during mitosis [2].
This proposal aims to test the hypothesis that the generation of positive superhelical stress is the in vivo basis for condensin's role in chromosome segregation and compaction. Using specific mutations in the condensin subunits we will correlate the positive supercoiling activity of condensin with endogenous chromosome segregation and also the extent of chromosome re-organisation induced by condensin during mitosis. Chromosome segregation will be assessed using GFP tagged chromosomal loci, DNA supercoiling by high-resolution agarose gel electrophoresis assays and chromosome structure by chromatin conformation capture techniques.
We will then use this data to build an in vitro system to model how condensin mediated positive supercoiling drives decatenation of intertwined mini-chromosomes by topoisomerase II.
Using this combination of in vivo and in vitro techniques the proposal aims to build a comprehensive model of how condensin changes chromosome structure and hence promotes faithful chromosomal inheritance and genome stability.


1. Hirano T: Annu Rev Biochem 2000, 69:115-144.
2. Baxter J, Sen N, Martinez VL, De Carandini ME, Schvartzman JB, Diffley JF, Aragon L: Science 2011, 331:1328-1332.

The principle impact of this proposal will be on basic research and clinical research into cancer therapeutics.
The mechanism of how condensin and SMC proteins in general maintain genomic stability is a crucial issue in the field of cell division. In particular the role of ATP hydrolysis in SMC function is an area of intense research. This proposal is aimed to be complementary to other studies into the role of SMC activity and may provide insights not observed by other approaches. Therefore the results of this work will be of widespread impact in the field.
This proposal could also impact on clinical cancer research. Condensin activity is known to be associated with topoisomerase II action. Topoisomerase II is the target of a number of chemotherapeutic agents. Understanding how condensin interacts with topoisomerase II within cells could potentially open up new avenues for how these drugs could be combined with other agents to maximise their efficacy. In addition there is preliminary evidence that condensin may be involved in the progression of certain types of cancer (see case for support and pathways to impact for further details). The pathways to impact document outlines how the proposed studies will be used to aid these clinical studies during the course of the proposal.
Within the immediate context of the department these studies will bring new technical expertise to the school but also a novel emphasis on the importance of DNA topology on DNA metabolism. In my experience the importance of DNA topological changes on chromosome dynamics is generally underappreciated. Through building a group that is focused on the importance of DNA topology on chromosome structure I hope to have an impact on the general thinking of other colleagues studying DNA metabolism. Since the science of DNA topology is interdisciplinary, with important physical research into polymer dynamics and mathematical work on topology, I also hope this work will foster collaborations between the different science departments on campus and generate novel insights for all concerned.