NSF-BSF: Defining the relationship between DNA replication kinetics and macromolecular protein assembly at the centromere

遗传信息的遗传需要DNA被复制并包装成一种带有分子“接头”或“手柄”的形式，从而使DNA能够有效地分离到子细胞中。然而，人们对这两个遗传步骤之间的关系知之甚少。该项目将为DNA复制协调及其在细胞分裂过程中随后分离的早期步骤奠定科学原理的基础。这项工作也将有助于对这些DNA代谢过程如何在活细胞内实时发生的新理解。该项目将为以色列和美国的研究生提供跨学科教育，交叉训练他们批判性地评估细胞生物学、数学、统计学和热力学方面的实验技术。为了进一步扩大该项目的影响，首席研究员和研究小组的成员将参与一些外展活动，向公众传达DNA遗传的复杂性和优雅性。PI Bloom将在凯南教师领导力研究员项目中担任导师，该项目是北卡罗来纳州一个成熟的STEM教师领导力项目。为了促进代表性不足的群体更广泛地参与科学，PI将指导由NC-Louis Stokes少数民族参与联盟（NC-LSAMP）赞助的科学和数学成就和足智多谋跟踪（SMART）计划。在真核细胞分裂过程中，着丝粒对着丝点组装和染色体分离至关重要。在每个细胞周期中，着丝粒DNA和着丝粒蛋白质的复合体必须被拆解以允许DNA复制，然后重新组装以允许微管附着和染色体分离。尽管进行了广泛的研究，但对于通过着丝粒区域的复制体进展与这些位点上功能性着丝粒的组装之间的相互作用知之甚少。本项目将结合遗传学、细胞生物学、活细胞显微镜和计算方法，研究有丝分裂过程中DNA复制、着丝粒建立、着丝粒组装和染色体分离之间的关系。具体来说，为了研究DNA结构对着丝粒组装的重要性，将使用计算机模拟和最近开发的活细胞成像方法来监测通过活跃和非活跃着丝粒区域的复制叉进程。这种综合方法适用于各种突变菌株和条件，将使该项目对着丝粒DNA复制、基因组稳定性、染色体分离和细胞分裂的不同方面有新的认识。这将为着丝粒区域的转录如何影响着丝粒组装提供关键的见解。这个美国/以色列合作项目由美国国家科学基金会和以色列两国科学基金会支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The inheritance of genetic information requires that DNA be replicated and packaged into a form with molecular "adaptors" or "handles" that allow the DNA to be efficiently segregated into daughter cells. However, little is known about the relationship between these two steps of inheritance. This project will lay the foundation of scientific principles governing the coordination of DNA replication and these early steps of its subsequent segregation during cell division. This work will also contribute new understandings of how these DNA metabolic processes occur in real-time within a living cell. The project will result in the interdisciplinary education of graduate students in Israel and the US, cross-training them to critically evaluate experimental techniques in cell biology, mathematics, statistics, and thermodynamics. To further broaden the impact of the project, the Principal Investigator and members of the research groups will contribute to several outreach events to convey the complexity and elegance of DNA inheritance to the general public. PI Bloom will mentor in the Kenan Fellows Program for Teacher leadership, a well-established STEM teacher leadership program in North Carolina. To facilitate broader participation of underrepresented groups in science, the PI will mentor in the Science and Math Achievement and Resourcefulness Track (SMART) program sponsored by the NC-Louis Stokes Alliance for Minority Participation (NC-LSAMP) in science. Centromeres are essential for kinetochore assembly and chromosome segregation during the eukaryotic cell division. At each cell cycle, the complex of centromere DNA and kinetochore proteins must be disassembled to allow DNA replication and then reassembled to allow microtubule attachment and chromosome segregation. Despite extensive studies, little is known regarding the interplay between replisome progression through centromeric regions and the assembly of functional kinetochores at these sites. This project will combine genetics, cell biology, live-cell microscopy, and computational methods to study the relationship between DNA replication, centromere establishment, kinetochore assembly, and chromosome segregation during mitosis. Specifically, to investigate the importance of DNA configurations to centromere assembly, computer simulations and recently developed live-cell imaging approaches for monitoring replication fork progression through active and inactive centromeric regions will be used. This integrative approach, applied to a variety of mutant strains and conditions, will enable the project to shed new light on different aspects of centromeric DNA replication, genome stability, chromosome segregation and cell division. This will provide critical insight into how transcription of centromeric regions affects kinetochore assembly.This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.