Development of novel single-molecule approaches for nanoscale study of helicases

描述(申请人提供)：单分子方法可用于实现对发生在纳米(Nm)尺度、毫秒(Ms)时间域和皮牛顿(Pn)力水平的生物过程的基本理解。在这项研究中，我们将开发单对荧光共振能量转移(SpFRET)、磁镊子(MT)和组合仪器的新用途，用于分析DNA结构的特定解旋酶，生命所必需的马达蛋白。我们将发展扫描共聚焦荧光显微镜/磁性镊子(SCFM/MT)来研究解旋酶和解旋酶-蛋白质复合体与DNA的相互作用，以及可以跟踪同一分子上两个染料对的spFRET的扫描共聚焦荧光显微镜(SCFM)。我们还将开发一种水平MT来跟踪直接在x和y平面上固定化的解旋酶活性以及DNA分子的旋转引起的扭曲限制的DNA分子的锚链长度的变化，以及新的spFRET分析来了解解旋酶的作用机制。我们将在这些研究中利用PcrA解旋酶。解旋酶在DNA和RNA代谢中起着关键作用，包括DNA复制、修复、重组和转录。革兰氏阳性菌的基因组编码一种基本的、保守的解旋酶PcrA，它参与DNA修复和质粒卷绕环复制，也参与迄今尚未确定的基本细胞过程。这项建议中描述的实验很可能导致新仪器的开发，能够结合MT和spFRET的优点，扩大SCFM跟踪双spFRET的能力，并扩大MT的使用，以跟踪DNA上的运动蛋白质产生的纵向和旋转运动。这些仪器不仅可以研究DNA解旋酶，还可以研究作用于核酸的其他马达蛋白质，如DNA和RNA聚合酶、DNA修复和重组因子、染色质重塑因子等。这里描述的实验还将增加我们对一组重要马达蛋白质的作用机制的了解，例如参与许多DNA交易的PcrA解旋酶。

DESCRIPTION (provided by applicant): Single-molecule approaches can be used to achieve a fundamental understanding of biological processes occurring at the nanometer (nm) scale, in the millisecond (ms) time domain and at the piconewton (pN) force level. In this study, we will develop novel uses of single-pair Fluorescence Resonance Energy Transfer (spFRET), Magnetic Tweezers (MT) and combination instruments for the analysis of DNA-structure specific helicases, essential motor proteins for life. We will develop combination scanning confocal fluorescence microscope/magnetic tweezers (SCFM/MT) to study interactions between helicase and helicase-protein complexes and DNA, and a scanning confocal fluorescence microscope (SCFM) that can follow spFRET of two dye pairs on the same molecule. We will also develop a horizontal MT to follow tether length changes of the torsionally constrained DNA molecule induced by the immobilized helicase activity directly in the x- and y- plane as well as rotation of the DNA molecule in real time, and novel spFRET assays to understand the mechanism of action of a helicase. We will utilize the PcrA helicase in these studies. Helicases play critical roles in DNA and RNA metabolism, including DNA replication, repair, recombination and transcription. The genomes of Gram-positive bacteria encode an essential, conserved helicase, PcrA, which is involved in DNA repair and in plasmid rolling-circle replication, and also in as yet unidentified essential cellular processes. The experiments described in this proposal are likely to lead to the development of new instruments capable of combining the strengths of MT and spFRET, expand the ability of a SCFM to follow double spFRET, arid expand the use of MT to follow longitudinal and rotational movements generated by a motor protein on DNA. These instruments will allow studies on not only DNA helicases but also other motor proteins that act on nucleic acids, such as DNA and RNA polymerases, DNA repair and recombination factors, chromatin remodeling factors, etc. The experiments described here will also increase our understanding of the mechanism of action of an important group of motor proteins as exemplified by the PcrA helicase which is involved in a number of DNA transactions.