Fundamental Biological Processes Under Torsion

项目摘要 双链DNA(DsDNA)的螺旋性质天生促进了 在复制和转录等基本过程中的扭转应力。特别是， 复制将不可避免地产生DNA超级卷曲，这可能会编织或缠绕子体 DNA链，形成前链烷。这种交织在一起，如果没有得到适当的解决，就会导致DNA 损伤、基因组不稳定和复制停滞。尽管它们对细胞的生存至关重要，但它们 问题非常复杂，对实验构成了巨大的障碍。因此，我们的 机械性的理解仍然明显受到限制。这里提出的工作的目的是 为了解决染色质的扭转机械性能在决定 拓扑僵局的形成和解决，以及这对 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 包括单一的和编织的染色质底物。由于人们对拓扑异构酶是如何 与染色质底物相互作用，Aim 2将开发一种新的方法来监测拓扑异构酶 活性，并直接应用这种方法来检测不同的拓扑异构酶 确定它们在超卷去除和基材偏好方面的有效性。这也将是 允许研究治疗药物如何影响拓扑异构酶活性。最后，《目标3》将 广泛检查全基因组复制产生的扭转和拓扑异构酶结合位点。 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--包括既定的方法和新颖的分析方法。拟议的研究将 证明染色质固有的机械性能在基本的 生物过程，并将对人类疾病的治疗产生深远影响 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.