This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In the budding yeast Saccharomyces cerevisiae, Dbf4p initiates DNA synthesis by activating and escorting Cdc7p to origins of replication. Dbf4p is subsequently targeted for destruction by the Anaphase Promoting Complex (APC/C) through D-box motifs, thus preventing reinitiation of DNA synthesis. Many known substrates containing D-box motifs are targeted by the APC/C through one of two necessary mediators, Cdc20p/Fizzy and Cdh1p. I am taking both a genetic and biochemical approach to understand the regulated proteolysis of Dbf4p and selected mitotic proteins in Saccharomyces cerevisiae. Confocal microscopy is being used with strains that harbor both Dbf4-RFP and Cdc20-GFP or Cdh1-GFP to establish localization of these proteins through the cell cycle. Two-hybrid assays are also underway to determine if physical interactions between Dbf4p and either Cdc20p or Cdh1p exist. Both deletion and mutational analyses will then be used to identify sites of direct interaction between Dbf4p and the mediator, and TAP-tagged versions of Dbf4p will be used to coimmunoprecipitate the complex in order to ascertain this interaction at the biochemical level. Additionally, synthetic genome analysis is being performed using a temperature sensitive allele of cdc20 in order to dissect genetic interactions. Characterization of the proteolysis of Dbf4p will help to elucidate the controls involved on the orderly progression of the cell cycle among eukaryotic organisms.
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