Roles of Sld2 and Sld3 in the Initiation of DNA Replication

智力优势：在这个项目中，生化分析和酵母遗传学将被用来了解DNA复制是如何在真核生物中开始的。该项目将重点研究DNA复制起始所需的两种蛋白质，Sld2和Sld3。Sld2和Sld3对DNA复制起始至关重要，但一旦DNA复制过程开始，这两种蛋白质就不参与复制。目前对Sld2和Sld3如何触发DNA复制的启动知之甚少。该研究项目将利用在PI实验室发现的Sld3与Mcm2-7复合物结合的观察结果，Mcm2-7复合物是一种在DNA复制过程中主动解开双螺旋DNA的蛋白质组装体。该研究项目还将利用PI实验室发现的Sld2和Sld3与DNA复制起源（即DNA复制开始的地方）的单链DNA结合的观察结果。这个研究项目将有助于我们了解真核生物中DNA复制是如何开始的。更广泛的影响：PI将与范德比尔特大学科学推广中心合作，向高中生教授DNA复制，该项目将面向来自代表性不足的背景和种族的高中生。课程单元将侧重于PI对出芽酵母蛋白Sld2和Sld3及其与DNA复制起始的关系的研究。在课程中，基本概念将涵盖如DNA结构，复制和测序。课程还将包括一个实验室，根据初步数据表明，Sld2与富含胸腺嘧啶的ssDNA结合，在体外随机选择带有Sld2的ssDNA。范德比尔特的外联人员将帮助实施该教学计划的绩效，该计划的评估将由外部评估人员进行。PI还将与范德比尔特大学的外联人员合作，将高中学生课程改编为地区教师的专业发展课程。这项工作将提高纳什维尔大都会地区高中生的科学素养。

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.