The Role of Tuberin and Cyclin B1 as a DNA Damage Response during the G2/M Transition

Tuberous Sclerosis (TS) is a multi-system disorder that causes the formation of benign tumours called hamartomas. In rare cases this disease can progress to aggressive cancers. The cause of TS is a mutation in either the TSC1 or TSC2 gene that encode for the tumour suppressor proteins Hamartin or Tuberin protein, respectively. Dr. Porter’s lab has characterized how Tuberin is able to regulate the G2/M transition of the cell cycle by binding and regulating the localization of CyclinB1, the protein that allows for the transition of the cell into mitosis. The hypothesis is that misregulation of this process may facilitate the accumulation of damaged DNA allowing for progression to malignancy. This thesis answers the following questions: What are the sites on CyclinB1 that are important in mediating the interaction between Tuberin? Does interaction with CyclinB1 stabilize the Tuberin protein? Does altered binding result in the accumulation of DNA damage? These questions will be assessed with 3 aims: 1) CyclinB1 mutants was constructed and binding in HEK293 has been measured using immunoprecipitation and western blotting techniques. 2) The stability of Tuberin was tested with graded amounts of CyclinB1 and measured using western blotting techniques. 3) The accumulation of DNA damage was tested with wild-type or mutated forms of Tuberin with reduced binding to CyclinB1. The data demonstrates that post-translational modifications of CyclinB1 are critical for mediating the interaction with Tuberin and that abrogation of this binding results in reduced protein levels of Tuberin and enhanced accumulation of DNA damage. These findings support that the Tuberin-CyclinB1 interaction is an important cellular checkpoint that protects cell integrity. Dissecting the mechanics of this checkpoint may reveal novel mechanisms for treating select benign and malignant tumours that impact the lives of many Canadians annually, with the majority of these being pediatric cases