Investigating the Role of Orcs in Chromatin Organization and in Mitotic Progression

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。智力价值：在多细胞生物体中，遗传物质的准确复制及其与子细胞的忠实分离是如何协调的，这是一个重要的问题，仍然需要清楚地理解。 在真核细胞中，这两个事件发生在交替的振荡周期中。DNA复制的起始需要多蛋白复合物的有序组装，包括在DNA复制起点处的起始识别复合物（ORC）。 在哺乳动物细胞中，染色体结构域占据特定的核位置，并在细胞分裂周期的S期期间的限定时间复制。染色质的变化和特定的修饰协调了ORC介导的DNA复制的时空分布的建立。 ORC蛋白不仅对DNA复制的起始至关重要，而且还涉及非preRC功能，包括异染色质组织和沉默，染色体分离和几种模式生物中的有丝分裂。本项目的目标是解决如何在哺乳动物细胞中协调DNA复制与染色质组织和有丝分裂进程。ORC亚基与复制起点、异染色质、着丝粒/动粒、中心体和细胞动力学沟的关联表明，ORC通过以细胞周期依赖的方式与不同的蛋白质伴侣相互作用，在协调染色体遗传周期的所有阶段中发挥关键作用。该项目将确定在细胞周期的不同阶段与ORC结合的新型蛋白质伴侣所发挥的作用，这将解决ORC如何参与各种细胞过程。特别是，该项目将提供重要的见解，在招聘和扩散的染色质蛋白质异染色质的ORC的作用。 最后，几种方法将被用来了解是否在染色质的组织和有丝分裂的作用是独立的，它在DNA复制启动的功能。由于ORC参与染色质组织是保守的从酵母到人类，这个项目的结果将有广泛的科学影响。更广泛的影响：这项研究是一个积极的推广计划，因为它利用了广泛的显微镜和活细胞成像，这是视觉上吸引人，并带来了细胞生物学的力量，在理解生物学的基本概念。在大学的设置提供了一个独特的机会，指导本科生，研究生和博士后研究员。该项目整合了生物化学，分子生物学和细胞生物学的使用，以吸引学生的兴趣，不仅来自生物学背景，而且来自化学和生物工程。该项目还将涉及为学生提供细胞和分子生物学概念的直接视图，包括积极指导本科生，包括妇女和少数民族。 该项目将使人们能够理解一个基本的生物学问题，即细胞如何完成DNA复制并发出信号使其进行染色体分离和细胞分裂。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual merit: How the accurate duplication of genetic material and its faithful segregation to daughter cells is coordinated in multi-cellular organisms is an important question that remains to be clearly understood. In eukaryotic cells these two events occur in alternative oscillating cycles. Initiation of DNA replication requires the ordered assembly of a multiprotein complex including the Origin Recognition Complex (ORC) at origins of DNA replication. In mammalian cells chromosomal domains occupy specific nuclear positions and replicate at defined times during S-phase of the cell division cycle. Chromatin changes and specific modifications orchestrate the establishment of spatio-temporal profiling of DNA replication that is ORC-mediated. ORC proteins are critical not only for initiation of DNA replication, but have also been implicated in non-preRC functions including heterochromatin organization and silencing, chromosome segregation and mitosis in several model organisms. The objective of this project is to address how ORCs coordinate DNA replication with chromatin organization and mitotic progression in mammalian cells. The association of ORC subunits with replication origins, heterochromatin, centromeres/kinetochores, centrosomes and cytokinetic furrow suggests that ORCs play a pivotal role in coordinating all stages of chromosome inheritance cycle by interacting with different protein partners in a cell-cycle dependent fashion. This project will identify the roles played by novel protein partners that bind to ORCs during different stages of the cell cycle that will address how ORC participates in various cellular processes. In particular this project will provide crucial insights into the role of ORCs in the recruitment and spreading of chromatin proteins to heterochromatin. Finally, several approaches will be used to understand whether the role of ORCs in chromatin organization and mitosis is independent of its function in DNA replication initiation. Since the involvement of ORCs in chromatin organization is conserved from yeast to humans the outcome of this project will have broad scientific impact.Broader impacts: This research is amenable to an active outreach program since it utilizes extensive microscopy and live cell imaging that is visually appealing and brings out the power of cell biology in understanding basic concepts of Biology. Being in a University setup gives a unique opportunity to mentor undergraduate, graduate students and postdoctoral fellows. This project integrates the use of Biochemistry, Molecular Biology and Cell Biology to capture the interest of students not only from Biology background but also from Chemistry and Bioengineering. This project will also involve providing students with a direct view of the concepts cell and molecular biology including actively mentoring undergraduate students, including women and minorities. This project will enable the understanding of a basic biology question, on how a cell accomplishes DNA replication and signals it to undergo chromosome segregation and cell division.