Roles of Sld2 and Sld3 in the Initiation of DNA Replication

智力价值：在这个项目中，将使用生化分析和酵母遗传学来了解DNA复制是如何在真核生物中启动的。该项目将重点研究启动DNA复制所需的两种蛋白质：SLD2和SLD3。SLD2和SLD3是DNA复制起始所必需的，但一旦DNA复制过程开始，这两种蛋白就不参与复制。目前对SLD2和SLD3如何启动DNA复制知之甚少。该研究项目将利用在PI的实验室中发现的SLD3与McM2-7复合体结合的观察结果，McM2-7复合体是一种蛋白质组件，在DNA复制过程中主动解开双螺旋DNA。该研究项目还将利用在PI的实验室中发现的SLD2和SLD3与DNA复制起点(即DNA复制开始处)发现的单链DNA结合的观察结果。这项研究项目将帮助我们了解DNA复制是如何在真核生物中启动的。广泛的影响：PI将与范德比尔特大学科学外展中心合作，向高中生教授DNA复制，这个项目将面向来自代表性不足的背景和种族的高中生。课程单元将集中于PI对发芽酵母蛋白SLD2和SLD3的研究，以及它们与DNA复制启动的关系。在课程中，将涵盖基本概念，如DNA结构、复制和测序。该课程还将包括一个实验室，根据初步数据显示SLD2与富含胸腺嘧啶的SSDNA结合，在体外选择带有SLD2的随机SSDNA。范德比尔特的外联工作人员将帮助实施这一教学计划的表现，该计划的评估将由外部评估员进行。PI还将与Vanderbilt的外联工作人员合作，将高中学生课程调整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市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.