The four specific aims of this proposal apply a multiplicity of strategies to elucidate the signal transduction pathways upstream of p53, and to determine which regions of the p53 molecule receive these signals. Mechanical microinjection of precisely defined DNA molecules will be used to study the cellular response to DNA damage; this will avoid complications introduced by radiation or drug treatments that activate signal response pathways unrelated to genomic injury. Recently described mutant cell lines with defects in the Ku protein that target a DNA dependent protein kinase to sites of DNA damage will be employed to assess whether it or the kinase are involved in the DNA damage or metabolite limitation responses. The potential involvement of p53 in the senescence clock will be analyzed using microinjection to introduce telomere-like molecules of different sizes and structures extrachromosomally. Alternatively rare cutting endonucleases will be used to generate similar structures de novo in chromosomes. The ability of mismatched bases to activate p53 will be analyzed by genetic strategies to render normal cells defective for mismatch repair, and microinjection to introduce mismatch substrates in vivo. Finally, structure-function relationships will be analyzed for the conserved protein kinase target sites in the N- and C-terminal regions of p53. This latter project will employ site specific recombinases to insure that each mutated p53 allele is analyzed in the same chromosomal site, and that all are expressed at equivalent and nearly wild type levels.
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