Roles of Sld2 and Sld3 in the Initiation of DNA Replication

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Intellectual Merit: In this project, biochemical assays and yeast genetics will be employed to understand how DNA replication is initiated in eukaryotes. This project will focus on two proteins that are required for the initiation of DNA replication, Sld2 and Sld3. Sld2 and Sld3 are essential for DNA replication initiation, but these two proteins are not involved in replication once the process of duplicating DNA has begun. Little is currently known about how Sld2 and Sld3 trigger the initiation of DNA replication. The research project will capitalize on observations discovered in the PI's lab that Sld3 binds to the Mcm2-7 complex, a protein assembly that actively unwinds double-helical DNA during the process of DNA replication. The research project will also capitalize on observations discovered in the PI's lab that Sld2 and Sld3 bind to single-stranded DNA that is found at the origin of DNA replication (i.e., where DNA replication starts). This research project will help our understanding of how DNA replication is initiated in a eukaryotic organism.Broader Impacts: The PI will work with the Vanderbilt University Center for Science Outreach to teach DNA replication to high school students, and this program will be for high school students from underrepresented backgrounds and ethnicities. The curriculum unit will focus on the PI's research on budding yeast proteins Sld2 and Sld3 and their relationship with initiation of DNA replication. In the curriculum, basic concepts will be covered such as DNA structure, replication, and sequencing. The curriculum will also include a laboratory with an in vitro selection of random ssDNA with Sld2, based upon preliminary data suggesting that Sld2 binds to thymine-rich ssDNA. Vanderbilt's Outreach staff will help implement the performance of this teaching program, and the evaluation of this program will be conducted by an external evaluator. The PI will also work with Vanderbilt's Outreach staff to adapt the high school student curriculum into a professional development curriculum for area teachers. This work will raise the scientific literacy of high school students in the Nashville Metropolitan area.