Title: Investigating the function of topoisomerase 2α in shaping mitotic chromosomes.
Outline: Type 2 topoisomerases catalyse the passage of one DNA duplex through another by formation of a transient double strand break in one of the duplexes. They are critical for maintaining a stable genome and are targets for many antiproliferative drugs. Mammals have two forms of topoisomerase 2, alpha and beta (topo 2α and β). Unlike the beta isoform, topo 2α is essential for the viability of dividing cells, since in its absence mitotic chromosome formation and segregation is lethally impaired. Topo 2α activity is vastly increased in late G2, and this is essential to allow the dramatic axial shortening of chromosomes that is required during M-phase (1,2). A potential regulator of topo 2α activity at this stage of the cell cycle is the prolyl isomerase Pin1. Disrupting Pin1 activity perturbs mitotic chromosome formation and influences the phosphorylation of topo 2α 3. A human cell line that is a conditional knock-out for topo 2α will be used to identify which of the ten putative Pin1-interacting motifs (S/Tp-P) in Topo 2α is required for its interaction with Pin1. The impact of various mutations on the protein’s phosphorylation, it’s interaction with Pin1 and on mitotic chromosome condensation and segregation will be assessed using immunoblotting, immunoprecipitation and fluorescence microscopy. This work will allow experience to be acquired in a range of wet-lab techniques, including molecular and protein biology (site-directed mutagenisis, cloning and plasmid construction, western blotting), mammalian tissue culture (including transfection protocols) and fluorescence microscopy (including indirect immunofluorescence).
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