Fundamental Biological Processes Under Torsion

项目摘要 双链DNA（dsDNA）的螺旋性质先天地促进了DNA的产生。 在复制和转录等基本过程中的扭转应力。特别是， 复制过程中不可避免地会产生DNA超螺旋，这种超螺旋可能会编织或插入子代 DNA链，产生前环烷烃。这种缠绕，如果没有得到适当的解决， 损伤、基因组不稳定性和复制停滞。虽然对细胞活力至关重要，但这些 这些问题非常复杂，给实验造成了重大障碍。因此我们的 对机械论的理解仍然非常有限。这里提出的工作旨在 为了解决染色质的扭转机械特性在决定细胞的结构中所起的作用， 拓扑僵局的形成和解决，反过来，这对 拓扑异构酶功能目标1将建立创建、基准测试和操作的方法 单个和编织染色质底物。由于我们对拓扑异构酶 与染色质底物相互作用，Aim 2将开发一种新的检测拓扑异构酶的方法 活动，在真实的时间，并直接应用这种方法来检查不同的拓扑异构酶， 确定它们在去除超螺旋和基底偏好方面的有效性。这也将 允许研究治疗剂如何影响拓扑异构酶活性。最后，Aim 3将 研究广泛复制产生的扭转和拓扑异构酶结合位点的基因组范围。 为了实现这些目标，我们将利用最先进的单分子和全基因组 技术-包括已建立的方法和新的测定。拟议的研究将 证明了染色质的内在机械特性在基础研究中的广泛作用。 将对人类疾病的治疗产生深远的影响 以及新型治疗剂的开发。

Project Summary The helical nature of double stranded DNA (dsDNA) innately promotes the generation of torsional stress during essential processes such as replication and transcription. In particular, replication will inevitably generate DNA supercoiling which may braid or intertwine daughter DNA strands, creating precatenanes. Such intertwining, if not properly resolved, results in DNA damage, genome instability, and replication arrest. Though critical to cellular viability, these problems are highly complex, posing significant barriers to experimentation. Thus our mechanistic understanding has remained conspicuously limited. The work proposed here aims to address the role that the torsional mechanical properties of chromatin play in determining the formation and resolution of topological impasses, and in turn, the implications this has for topoisomerase function. Aim 1 will establish methods to create, benchmark, and manipulate both single and braided chromatin substrates. Since little is known about how topoisomerases interact with chromatin substrates, Aim 2 will develop a novel assay to monitor topoisomerase activity, in real time, and directly apply this approach to examine different topoisomerases to determine their effectiveness in supercoiling removal and substrate preferences. This will also allow investigation into how therapeutic agents impact topoisomerase activity. Finally, Aim 3 will examine broadly replication generated torsion and topoisomerase binding sites genome wide. To pursue these aims we will leverage state-of-the-art single molecule and genome wide techniques – including established approaches and novel assays. The proposed research will demonstrate the broad role of the intrinsic mechanical properties of chromatin in fundamental biological processes and will have far-reaching impacts into the treatment of human disease and the development of novel therapeutic agents.