We are exploiting the Center's array of technologies to study a complex physiological process, the yeast DNA damage response, in a systematic and comprehensive manner in order to define the network of interactions that occur among components of the DNA replication, repair, and cell cycle machineries. Our objectives in year 2 were two-fold: First, to develop a rapid and robust method of protein purification that permits high-throughput characterization of yeast protein complexes. Second, to use a model cellular process upon which to both evaluate the effectiveness of the global approaches employed at the Center and to refine the Center's ability to generate, integrate, and disseminate biological data. Finally, we have developed a working set of guidelines for collaboration with the Center. Our principal method of choice to study the molecular interactions that mediate the DNA damage response is to use MS/MS spectrometry to characterize the composition of protein complexes isolated by affinity chromatography. Currently, we have tagged over 40 key damage response proteins and have performed affinity-purification on most of these. Preliminary characterization by MS/MS has revealed a number of novel protein-protein interactions.
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