Characterization of Dbf4/Cdc7 function in DNA replication and cell cycle checkpoint responses

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We live in a time of uncertainty when changes to the environment as a consequence of increased industrial activities pose new risks to all living organisms. Among the most concerning of these is an increased incidence of cellular perturbations. For example, exposure to common manufacturing chemical benzene or to certain types of commercial nanoparticles can lead to DNA damage and genomic instability. My lab is studying the way that cells normally copy their DNA as they divide. Detailed knowledge of how this occurs under normal conditions helps us to understand how things go wrong and leads to new strategies to help prevent harmful outcomes. One of the safeguards cells use to protect their genomes is a surveillance system called cell cycle checkpoints. They monitor the presence of DNA damage, coordinate repair processes when necessary, and eliminate severely damaged cells from the population if they cannot be efficiently restored. We are particularly interested in a key protein called Dbf4, found in all eukaryotic (with a clearly defined nucleus) organisms, which is both required for proper DNA replication to initiate, and is inhibited by checkpoints in response to DNA damage as a means of slowing down DNA replication until the damage has been repaired. The three objectives of my research proposal help us to understand how Dbf4 functions and is regulated. All of the work will be carried out in budding yeast cells, a very useful model organism, with most cellular functions similar to that of other eukaryotic organisms, including humans. For Objective 1 we are proposing to further characterize a newly identified way that a key checkpoint protein, Rad53, interacts with Dbf4. We are interested in determining whether this is specific to the Dbf4-Rad53 interaction, or if it may operate in other interactions involving checkpoint proteins. For Objective 2 we will study how Dbf4 interacts with and opposes the activity a protein called Rif1 which acts as an inhibitor of DNA replication. Of particular interest is a better characterization of the effects of disrupting the Dbf4-Rif1 association, which causes cells to become acutely sensitive to DNA double strand breaks which can occur when cells are exposed to environmental carcinogens. For Objective 3 we will further characterize the interaction between Dbf4 and a protein called Mrc1, which appears to be important for the proper timing of DNA replication. The students involved in this research will benefit from training in a wide range of approaches that they will employ to investigate these aspects of Dbf4 activity and regulation, including biochemistry, molecular biology, genetics, structural analysis and bioinformatic techniques.