Enhancement of Cellular DNA Flexibility

描述(由申请人提供)：我们的实验室对了解DNA僵硬的物理来源和生物管理感兴趣。长期以来，人们一直认为双链DNA是所有天然聚合物中最坚硬的一种。值得注意的是，经过50年的研究，DNA僵硬的来源仍然不清楚。更好地了解DNA内部的力平衡将揭示DNA弯曲蛋白(如组蛋白八聚体)克服的力，并可能控制纳米材料和纳米设备的DNA硬度。活细胞是如何管理僵硬的DNA聚合物的，机制细节也不清楚。DNA在细胞内的弯曲和扭转弹性似乎比在溶液中更高。为什么？我们一直在研究序列非特定的HMGB DNA弯曲蛋白和其他“结构”蛋白，以此作为模型来理解DNA可能被赋予细胞内增强的表观灵活性的一种机制。了解和利用这些蛋白质的性质可以通过靶向蛋白质来调节转录调控所需的DNA环的稳定性，从而在人工基因控制中得到应用。在之前的资助期间，我们在了解DNA僵硬的来源和管理方面取得了重要进展。我们现在提出四个目标来继续这一基础研究。目的1确定DNA电荷密度、碱基堆积和DNA刚性之间的关系。目的2体外检测HMGB蛋白结合对DNA和染色质柔韧性和结构的影响。目的3将加深我们对大肠杆菌中建筑蛋白增强DNA环的基础的理解。最后，目标4将测量HMGB蛋白对酵母中DNA环的影响。 与公共健康相关：DNA分子包含所有生物的信息代码。这种信息包含在非常长的双螺旋DNA分子中。从远处看，这些分子是线状的，但从必须结合和阅读DNA的蛋白质的角度来看，它们是棒状的。这项更新资金的提议将使我们由分子生物学家、生物化学家和物理学家组成的合作研究小组继续我们的生产性项目，以了解为什么DNA在当地是僵硬的和杆状的，以及一种被称为“建筑蛋白”的特殊蛋白质如何通过引起弯曲和扭结来增加DNA的灵活性。

DESCRIPTION (provided by applicant): Our laboratory is interested in understanding the physical origin and biological management of DNA stiffness. It has long been appreciated that duplex DNA is among the stiffest of all natural polymers. Remarkably, the origin of DNA stiffness remains obscure after 50 years of study. A better understanding of the balance of forces within DNA would reveal what forces are overcome by DNA bending proteins such as histone octamers, and could allow control of DNA stiffness for nanomaterials and nanodevices. How living cells manage the stiff DNA polymer is also not understood in mechanistic detail. The bending and twisting flexibility of DNA within cells appears to be higher than in solution. Why? We have been studying sequence non-specific HMGB DNA bending proteins and other "architectural" proteins as models for understanding one mechanism by which DNA might be endowed with enhanced apparent flexibility in cells. Understanding and harnessing the properties of these proteins could have applications in artificial gene control by targeting the proteins to modulate the stability of DNA loops required for transcriptional regulation. During the previous funding period we made important progress toward understanding the origin and management of DNA stiffness. We now propose four aims to continue this fundamental research. Aim 1 will determine the relationship between DNA charge density, base stacking and DNA stiffness. Aim 2 will measure effects of HMGB protein binding on the flexibility and structure of DNA and chromatin in vitro. Aim 3 will improve our understanding of the basis for DNA looping enhancement by architectural proteins in E. coli. Finally, Aim 4 will measure the effects of HMGB proteins on DNA looping in yeast. PUBLIC HEALTH RELEVANCE: DNA molecules contain the information code for all living things. This information is contained in very long double-helix DNA molecules. These molecules are thread-like when considered at a distance, but are rod-like from the perspective of the proteins that must bind and read DNA. This proposal for renewed funding will allow our collaborative research group of molecular biologists, biochemists, and physicists to continue our productive projects to understand why DNA is stiff and rod-like locally, and how a special group of proteins called "architectural proteins" increase the flexibility of DNA by causing bends and kinks.