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：在多细胞生物中，遗传物质的精确复制及其对子细胞的忠实分离是如何协调的，这是一个仍有待明确理解的重要问题。在真核细胞中，这两个事件以交替的振荡周期发生。DNA复制的起始需要多蛋白复合体的有序组装，包括DNA复制起始处的起源识别复合体（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期的特定时间复制。染色质变化和特异性修饰协调了orc介导的DNA复制时空谱的建立。ORC蛋白不仅对DNA复制的起始至关重要，而且在一些模式生物中也涉及非prerc功能，包括异染色质组织和沉默，染色体分离和有丝分裂。该项目的目的是解决ORCs如何协调DNA复制与染色质组织和有丝分裂进展在哺乳动物细胞。ORC亚基与复制起点、异染色质、着丝粒/着丝点、中心体和细胞动力学沟的关联表明，ORC通过与不同的蛋白质伴侣以细胞周期依赖的方式相互作用，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起的作用，这将解决ORC如何参与各种细胞过程。特别是，该项目将为ORCs在染色质蛋白向异染色质的募集和扩散中的作用提供至关重要的见解。最后，将使用几种方法来了解ORCs在染色质组织和有丝分裂中的作用是否独立于其在DNA复制起始中的功能。由于ORCs在染色质组织中的参与从酵母到人类都是保守的，因此该项目的结果将具有广泛的科学影响。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，这在视觉上很有吸引力，并在理解生物学的基本概念方面展现了细胞生物学的力量。在大学的设置提供了一个独特的机会来指导本科生，研究生和博士后。本项目整合了生物化学、分子生物学和细胞生物学的应用，既吸引了生物学背景的学生，也吸引了化学和生物工程专业的学生。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。这个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复制并发出信号以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.