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.

该研究小组将研究特定染色体结构域的生物物理特性，以及它们在整个细胞周期中的作用。DNA和RNA聚合酶在复制和转录过程中产生相当大的力（高达40 pN）。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。复制染色体的姐妹着丝粒之间微管附着（~ 20 pN/微管）产生的张力对于确保后期开始时染色体分离的保真度的机制至关重要。因此，机械力在DNA代谢过程中起着至关重要的作用。然而，过大的力（10 pN）可以抑制S期染色质的组装和有丝分裂中具有两个着丝粒的染色体（双着丝粒染色体）的断裂。因此，在整个细胞周期中，染色体上的力可能在空间上和时间上都受到调节。最近的实验已经解决了在体外拉伸DNA和置换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进入核小体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。他们将对染色质施加力，以测量着丝粒、常染色质和端粒序列的力-延伸关系。智力优势：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结构域的生物物理特性的贡献。此外，他们还发现了在张力下识别DNA的蛋白质。通过检查缺乏这些组分的细胞中特定染色质结构域的力延伸曲线，他们将建立特定力延伸签名的遗传要求。他们希望这项工作能为整个真核染色体绘制出原力延伸图。这种方法将提供染色体的第一个生物力学观点，并将在理解能量和结构信息是如何存储的关键。更广泛的影响：这项研究将通过三个地点整合到教育和推广中：K12学生的网络分子操作项目，通过整合到本科科学视角课程中，以及通过广泛的本科研究计划。第一个程序允许K12学生在位于远程的AFM下操纵真实的分子（DNA，病毒）。在一个典型的一年，该计划达到200多名K12学生，让本科生和研究生研究人员的指导和令人兴奋的K12学生的经验。有丝分裂中的力量科学将作为科学观点课的一部分，“事物如何运作”，每年向250多名非科学专业的学生讲授。对于本科研究，教育目标将侧重于整合研究和教学活动，为学生提供评估和采用新技术的工具。 大约6名学生将在赠款的过程中在一个密集的研究为基础的计划中研究有丝分裂的力量。