Biomechanics of Chromosome Structure and Dynamics In Living Cells

研究小组将研究特定染色体区域的生物物理性质，以及在整个细胞周期中力在其功能中所起的作用。DNA和RNA聚合酶在复制和转录过程中产生相当大的力(高达40pN)。在有丝分裂过程中，微管附着在着丝粒区域，为染色体分离提供动力。复制染色体姐妹着丝粒之间的微管附着(~20pN/微管)所产生的张力对于确保后期染色体分离的保真度是至关重要的。因此，机械力在DNA代谢过程中起着至关重要的作用。然而，过大的外力(10pn)会抑制染色质在S期的组装和有丝分裂中双着丝粒染色体(双着丝粒)的断裂。因此，在整个细胞周期中，染色体上的作用力很可能在空间上和时间上都受到调节。最近的实验解决了在体外拉伸DNA和移位核小体所需的力的大小。这些实验揭示了核小体核心周围不同的DNA-蛋白质相互作用，并增强了我们对需要获得核小体DNA的酶过程的理解。在这个项目中，他们将分离特定的染色质结构域，并确定染色体不同区域的生物物理性质。他们将对染色质施加力，以测量着丝粒、常染色质和端粒序列的力-伸展关系。智力价值：使用这种方法，他们将剖析DNA序列和蛋白质结构对特定亚染色体区域的生物物理性质的贡献。此外，他们还确定了在紧张状态下识别DNA的蛋白质。通过检查细胞中缺乏这些成分的特定染色质区域的力延伸曲线，他们将建立特定力延伸特征的遗传要求。他们希望这项工作能为整个真核细胞的染色体绘制一张力延伸图。这种方法将提供对染色体的第一个生物力学观点，并将对理解能量和结构信息是如何存储的至关重要。更广泛的影响：这项研究将通过三个场所整合到教育和推广中：面向K12学生的联网分子操纵项目，通过融入本科科学视角课程，以及通过广泛的本科研究计划。第一个项目允许K12的学生在北卡罗来纳大学远程安装的原子力显微镜下操纵真实的分子(DNA、病毒)。在典型的一年中，该项目覆盖200多名K12学生，使本科生和研究生能够体验到指导和令人兴奋的K12学生的经历。有丝分裂中力的科学将作为科学视角课程的一部分，每年向250多名非理科专业的学生讲授《事物是如何工作的》。对于本科生的研究，教育目标将侧重于将研究和教学活动相结合，为学生提供评估和应用新技术的工具。在整个拨款过程中，大约6名学生将在一个密集的基于研究的项目中研究有丝分裂中的力量。

The research team will study the biophysical properties of specific chromosomal domains and the role forces play in their function throughout the cell cycle. DNA and RNA polymerases generate considerable force (up to 40pN) during processes of replication and transcription. During mitosis, microtubules attach to centromeric regions to provide the motive force for chromosome segregation. The tension generated by microtubule attachment (~20pN/microtubule) between sister centromeres of replicated chromosomes is critical to the mechanisms that ensure the fidelity of chromosome segregation upon anaphase onset. Thus mechanical force plays a critical role in DNA metabolic processes. However, excessive force (10pN) can inhibit chromatin assembly in S-phase and breakage of chromosomes with two centromeres (dicentric chromosomes) in mitosis. It is therefore likely that forces on chromosomes are spatially as well as temporally regulated throughout the cell cycle. Recent experiments have addressed the amount of force required to stretch DNA and displace nucleosomes in vitro. These experiments reveal different DNA-protein interactions around the nucleosome core and enhance our understanding of the enzymatic processes that require access to nucleosomal DNA. In this project they will isolate specific chromatin domains and determine the biophysical properties of distinct regions of the chromosomes. They will apply force to chromatin to measure the force-extension relationships for centromeres, euchromatin and telomeric sequences. Intellectual Merit: Using this approach, they will dissect the DNA sequence and protein structural contributions to the biophysical properties of specific sub-chromosomal domains. In addition, they have identified proteins that recognize DNA under tension. By examining the force extension curves for specific chromatin domains in cells lacking these components they will establish the genetic requirements for specific force extension signatures. They expect this work to lead to a Force-extension map for an entire eukaryotic chromosome. This approach will provide the first biomechanical view of the chromosome and will be critical in understanding how energy and structural information is stored. Broader Impact: This research will be integrated into education and outreach through three venues: a networked molecular manipulation project to K12 students, through integration into an undergraduate science perspective course, and through an extensive undergraduate research program. The first program allows K12 students to manipulate real molecules (DNA, Viruses) under an AFM that is located remotely at UNC. In a typical year this program reaches over 200 K12 students, allowing undergraduates and graduate researchers the experience of mentoring and exciting K12 students. The science of forces in mitosis will be included as a section in a science perspective class, "How Things Work" taught to over 250 non-science majors each year. For undergraduate research, educational goals will focus on integrating research and teaching activities to give students the tools to evaluate and employ new technologies. About 6 students will study forces in mitosis within an intensive research-based program over the course of the grant.