Single Molecule Studies of DNA 'Breathing' Fluctuations and their Roles in Helica

描述（由申请人提供）：细胞机器准确复制和转录DNA的能力对生物体的健康功能至关重要。虽然参与这些过程的成分在很大程度上是已知的，但这些成分发挥作用的分子机制尚不清楚，因此，导致复制或转录机制故障的过程也知之甚少。许多疾病都是这种功能失调的直接结果，包括某些形式的癌症，因此，对这些系统如何工作的更好理解可能最终导致迄今为止仍然难以捉摸的疾病的治愈。双链DNA“呼吸”是一种现象，指的是由于热波动导致双链DNA中氢键的短暂断裂和/或相邻核酸碱基的解开。由这些波动引起的DNA呼吸运动可能是基因组调控蛋白识别特定碱基序列机制的重要组成部分，包括在DNA复制和转录过程中催化双链DNA解绕的复制解旋酶。在提出的研究中，我们计划使用新的单分子光谱方法来观察和表征DNA呼吸过程中DNA碱基的长时间（微到毫秒）热波动。我们将利用固定在双链DNA构建体上的可见波长荧光探针直接观察DNA主链的波动，而利用荧光DNA碱基类似物标记的DNA构建体直接观察DNA碱基的波动。DNA呼吸波动在复制叉结附近的碱基应该大大增强，这应该在单分子水平上通过这些测量得到证实。随后的目标是将噬菌体T4编码的原体解旋酶引入这些DNA复制叉结构中，并观察解旋酶的存在和功能如何影响DNA呼吸波动。在这个提议中，假设DNA呼吸波动是在复制叉上解旋酶催化的dsDNA解绕的核心。如果这个假设是正确的，那么当解旋酶存在时，人们可能会看到DNA呼吸波动的显著变化。DNA呼吸和解旋酶功能相互依赖的表征将是理解驱动DNA复制和转录机制的重要一步。

DESCRIPTION (provided by applicant): The ability of the cellular machinery to accurately replicate and transcribe DNA is crucial to the healthy function of living organisms. While the components involved in these processes are largely known, the molecular mechanisms by which the components function are not well understood and, as a consequence, processes that result in malfunctions of the replication or transcription machinery are also poorly understood. Many diseases are a direct result of such malfunctions, including certain forms of cancer, and thus a better understanding of how these systems work may ultimately lead to cures for diseases which have heretofore remained elusive. Doubled-stranded (ds) DNA 'breathing' is a phenomenon that refers to the transient breaking of hydrogen-bonds and/or the unstacking of adjacent nucleic acid bases in dsDNA as a consequence of thermal fluctuations. The DNA breathing motions that result from these fluctuations are likely to be important components of the recognition mechanisms of specific base sequences by genome-regulatory proteins, including the replication helicases that catalyze the unwinding of double-stranded DNA during the processes of DNA replication and transcription. In the studies proposed we plan to use new single molecule spectroscopic approaches to observe and characterize the longer-lived (micro- to milliseconds) thermal fluctuations of DNA bases during the DNA breathing process. We will utilize visible wavelength fluorescent probes rigidly attached to double-stranded DNA constructs to directly observe fluctuations of the DNA backbone, while DNA constructs labeled with fluorescent DNA base analogues will be utilized to directly observe fluctuations of DNA bases. DNA breathing fluctuations should be greatly enhanced for bases in the vicinity of a replication fork junction, which should be confirmed at the single-molecule level by these measurements. A subsequent aim is to introduce the primosome helicase coded by bacteriophage T4 to these DNA replication fork constructs and observe how the DNA breathing fluctuations are affected by the presence and function of the helicase. It is hypothesized in this proposal that DNA breathing fluctuations are central to the helicase-catalyzed unwinding of dsDNA at a replication fork. If thi hypothesis is true, then one might expect to see significant changes in DNA breathing fluctuations when the helicase is present. The characterization of the interdependence of DNA breathing and helicase function will be a major step in the understanding of the mechanisms that drive DNA replication and transcription.