The p53 tumor suppressor invokes a cell cycle arrest in response to as few as one DNA double strand break. I will analyze upstream activators and downstream responders of p53 using a multifaceted approach involving microinjection of defined DNA substrates, structure-function analysis of mutated p53, and analysis of cell lines with defects in potential signal transducers. I will examine the role of Ku/DNA-PK as an upstream activator of p53 using microinjection of DNA substrates. I will complement these studies by developing cell lines with N- and C- terminal p53 mutations by site-specific recombination. This method will allow me to study p53 mutations expressed at normal levels in the cell and assess the role of various domains and canonical phosphorylation sites in the extremely sensitive p53-dependent response. In addition, shortened telomeres may act like broken chromosomes to invoke a p53-dependent arrest, linking p53 to the senescence clock. Therefore, I will create telomere substrates of different lengths and structures to microinject into p53+ and p53- cell lines and assay for their role in a p53-mediated response. As the pathway controlled by p53 is the most frequently rendered defective in cancer cells, we anticipate that an understanding of p53 function will help in the development of more rational chemotherapeutic agents.
