Activities of nucleoprotein complexes visualized in single-molecule experiments

项目摘要 对生物的机械论理解需要我们理解蛋白质和DNA是如何相互作用的 共同产生功能染色体。这种理解是保护人类健康的核心， 处理遗传疾病，与病原体作斗争。建议的项目主要集中在 单分子分析，允许直接可视化生物分子相互作用，并可用于 详细分析蛋白质与DNA的相互作用。该提案的目的包括仔细研究 从DNA中去除蛋白质的解离“交换”机制，这表明 对DNA上蛋白质周转的常规描述，这将影响广泛的基因研究 调节和染色体结构蛋白。第二个和第三个目标集中在研究 大的“染色体结构维持”(SMC)蛋白质复合体的作用机制 真核细胞中染色体的折叠。第二个目标涉及到理解 在分离中起关键作用的Smc5/6复合体的致密作用和DNA底物的选择 在细胞分裂过程中相互复制染色体，而第三个目的是直接显微镜观察 Smc5/6致密DNA机制的观察。第二和第三个目标还将包括 Smc5/6和更好理解的凝聚素复合体的并列比较。高度的 提出的DNA加工机制的机理分析将使我们更好地理解 细胞如何解释、折叠和改变它们的基因组，从而更好地理解 这些功能受损，更好地理解如何在致病性疾病中靶向这些功能 有机体。

Project Summary Mechanistic understanding of living things requires our understanding of how proteins and DNA interact together to generate functional chromosomes. This understanding is central to preserving human health, dealing with genetic disorders, and fighting pathogenic organisms. The proposed projects are focused on single-molecule analyses which permit direct visualization of biomolecule interactions, and can be used to analyze protein-DNA interactions in detail. The aims of the proposal include careful study of a facilitated dissociation "exchange" mechanism for removal of proteins from DNA that suggests a major revision of conventional descriptions of protein turnover on DNA, and which will affect a wide range of studies of gene regulatory and chromosome structural proteins. The second and third aims are focused on study of mechanisms of large "Structural Maintenance of Chromosomes" (SMC) protein complexes which mediate the folding of chromosomes in eukaryote cells. The second aim concerns understanding the mechanics of compaction and DNA substrate selection by the Smc5/6 complex, which plays a key role in separating replicated chromosomes from one another during cell division, while the third aim concerns direct microscopy observation of the mechanism by which Smc5/6 compacts DNA. The second and third aim will also include side-by-side comparisons of Smc5/6 and the more well-understood condensin complex. The highly mechanistic analyses of DNA processing machinery that are proposed will give us a stronger understanding of how cells interpret, fold and change their genomes, leading to a better understanding of pathologies where those functions are impaired, and better understanding of how to target those functions in pathogenic organisms.