Biomechanics of Chromosome Structure and Dynamics In Living Cells

活细胞染色体结构和动力学的生物力学

• 批准号: 0451240
• 负责人: Kerry Bloom
• 金额: $ 40.33万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0451240&HistoricalAwards=false
• 关键词: Biomechanics; Chromosome; Structure; Dynamics; Living

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.

研究小组将研究特定染色体结构域的生物物理特性，并在整个细胞周期中扮演其功能中的角色。在复制和转录过程中，DNA和RNA聚合酶产生相当大的力（高达40pn）。在有丝分裂期间，微管附着在丝粒区域上，以提供染色体分离的动力。微管附着（〜20pn/微管）在复制染色体的姐妹丝粒之间产生的张力对于确保染色体隔离在过后生开始的机制至关重要。因此，机械力在DNA代谢过程中起关键作用。但是，过量的力（10pn）可以抑制S期中的染色质组装，并在有丝分裂中使用两个centromeres（二含染色体）的染色体破裂。因此，在整个细胞周期中，染色体上的力在空间上和时间调节。最近的实验已经解决了在体外伸展DNA并置换核小体所需的力量。这些实验揭示了围绕核小体核心的不同DNA-蛋白质相互作用，并增强了我们对需要访问核小体DNA的酶促过程的理解。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特性。他们将向染色质施加力，以测量着丝粒，白染色质和端粒序列的力延伸关系。智力优点：使用这种方法，他们将剖析特定亚染色体域的生物物理特性的DNA序列和蛋白质结构贡献。此外，他们还确定了识别张力下DNA的蛋白质。通过检查缺乏这些成分的细胞中特定染色质结构域的力延伸曲线，它们将确定特定力扩展特征的遗传要求。他们希望这项工作会导致整个真核染色体的力延伸图。这种方法将提供染色体的第一个生物力学观点，对于理解如何存储能量和结构信息至关重要。更广泛的影响：这项研究将通过三个场所纳入教育和宣传：通过将K12学生的网络分子操纵项目集成到本科科学观点课程，并通过广泛的本科研究计划。第一个程序允许K12学生在AFM下操纵真实分子（DNA，病毒），该AFM位于UNC。在典型的一年中，该计划将吸引200多名K12学生，允许本科生和研究生研究人员获得指导和令人兴奋的K12学生的经验。有丝分裂的力学科学将作为科学视角的一部分，每年教授250多个非科学专业的“事物工作”。对于本科研究，教育目标将集中于整合研究和教学活动，以为学生提供评估和采用新技术的工具。 在赠款过程中，大约6名学生将在一个基于研究的计划中学习有丝分裂的力量。