Chromatin and Cell Cycle Regulation of ORC Function

描述(申请人提供)：本项目的总体目标是阐明调控酿酒酵母DNA复制起点活性的分子机制。起源识别复合体(Origin Recognition Complex，ORC)通过参与复制染色体DNA的复制复合体的组装，直接与真核复制起始点结合并调节其功能。起始复合体的组装和激活受细胞周期和检查点的调控，以确保有丝分裂分离前染色体复制的准确性。染色质结构也通过未知的机制调节起源功能。我们的指导性假设是，ORC与复制起始点相互作用的调节是后续起始点活动的关键决定因素。利用细胞、生化和遗传分析，我们将描述ORC与酿酒酵母复制起始点的细胞周期相互作用，重点研究染色质环境如何影响ORC-起始点的相互作用和起始点功能。本研究的具体目的是：1)确定ORC-起源结合的特征如何调节每个起源的活性，2)通过分析不同调控起源的组蛋白的结构修改来阐明DNA复制和染色质结构之间的关系，以及3)表征整个基因组中的这些相互作用。 对复制起点活性的严格调控确保了基因组向子代细胞传递的准确性，对细胞增殖的适当调节，以及细胞复制与基因表达和细胞分化的整合。了解复制起源活动在所有背景下是如何调节的，有可能为癌症的检测和治疗提供改进的方法，以及阐明发展机制。

DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate the molecular mechanisms that regulate the activity of DNA replication origins in S. cerevisiae. The origin recognition complex (ORC) directly binds to and regulates the function of eukaryotic replication origins by participating in the assembly of replication complexes that duplicate the chromosomal DNA. Assembly and activation of origin complexes is regulated by the cell cycle and checkpoints to ensure the accuracy of chromosome duplication prior to mitotic segregation. Chromatin structure also regulates origin function through unknown mechanisms. Our guiding hypothesis is that regulation of the interactions of ORC with replication origins is a critical determinant of subsequent origin activity. Using cellular, biochemical, and genetic analyses, we will characterize the cell cycle interactions of ORC with replication origins in S. cerevisiae, focussing on how the chromatin environment influences ORC-origin interactions and origin function. The specific aims of this research arc to: 1) determine how the characteristics of ORC-origin binding regulate the activity of each origin, 2) elucidate the relationship between DNA replication and chromatin structure by analyzing the structural modifications of histones at differently regulated origins, and 3) characterize these interactions throughout the genome. Strict regulation of the activity of replication origins ensures accuracy in the transmission of the genome to daughter cells, proper regulation of cellular proliferation, and integration of cell duplication with gene expression and cellular differentiation. Understanding how replication origin activity is regulated in all of its contexts has the potential of providing improved methods for the detection and treatment of cancer, as well as elucidating mechanisms of development.