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

遗传信息的遗传要求DNA被复制并包装成具有分子“接头”或“手柄”的形式，使DNA能够有效地分离到子细胞中。然而，人们对这两个继承步骤之间的关系知之甚少。该项目将为协调DNA复制及其在细胞分裂过程中后续分离的这些早期步骤奠定科学原理的基础。这项工作还将有助于对这些DNA代谢过程如何在活细胞内实时发生的新理解。该项目将对以色列和美国的研究生进行跨学科教育，交叉培训他们对细胞生物学、数学、统计学和热力学的实验技术进行批判性评估。为了进一步扩大该项目的影响，首席调查员和研究小组成员将参与几个外联活动，向公众传达DNA遗传的复杂性和高雅。皮布鲁姆将在凯南教师领导力研究员计划中担任导师，这是北卡罗来纳州一个久负盛名的STEM教师领导力计划。为了促进代表不足的群体更广泛地参与科学，国际科学协会将指导由北卡罗来纳州-路易斯·斯托克斯少数群体参与科学联盟(NC-LSAMP)赞助的科学和数学成就和足智多谋(SMART)计划。着丝粒是真核细胞分裂过程中着丝粒组装和染色体分离所必需的。在每个细胞周期，着丝粒DNA和动粒蛋白的复合体必须被分解以允许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，但对通过着丝粒区域的复制体进展与这些部位的功能动粒组装之间的相互作用知之甚少。这个项目将结合遗传学、细胞生物学、活细胞显微镜和计算方法来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。具体地说，为了研究DNA构型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，通过活跃和非活跃的着丝粒区域来监测复制分叉的进展。这一综合方法应用于各种突变菌株和条件，将使该项目能够为着丝粒DNA复制、基因组稳定性、染色体分离和细胞分裂的不同方面提供新的线索。这将提供对着丝粒区域转录如何影响着丝粒组装的关键洞察。这项美国/以色列合作项目得到了美国国家科学基金会和以色列双国科学基金会的支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.