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

描述（由申请人提供）：单分子方法可用于实现在纳米（nm）尺度，毫秒（ms）时域和皮牛顿（pN）力水平上发生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结构特异性解旋酶，生命必需的运动蛋白。我们将开发用于研究解旋酶和解旋酶蛋白复合物与DNA相互作用的扫描共聚焦荧光显微镜（SCFM/MT），以及用于跟踪同一分子上两对染料的spFRET的扫描共聚焦荧光显微镜（SCFM）。我们还将开发水平MT，以实时跟踪固定解旋酶活性在x和y平面上引起的扭转约束DNA分子的系链长度变化以及DNA分子的旋转，并开发新的spFRET测定方法以了解解旋酶的作用机制。我们将在这些研究中使用PcrA解旋酶。解旋酶在DNA和RNA的代谢过程中起着关键作用，包括DNA的复制、修复、重组和转录。革兰氏阳性菌的基因组编码一种重要的、保守的解旋酶，即PcrA，它参与DNA修复和质粒滚环复制，也参与尚未确定的基本细胞过程。本提案中描述的实验可能会导致能够结合MT和spFRET优势的新仪器的开发，扩展SCFM跟踪双spFRET的能力，并扩展MT的使用，以跟踪DNA上的马达蛋白产生的纵向和旋转运动。这些仪器不仅可以研究DNA解旋酶，还可以研究其他作用于核酸的运动蛋白，如DNA和RNA聚合酶、DNA修复和重组因子、染色质重塑因子等。这里描述的实验也将增加我们对一组重要运动蛋白的作用机制的理解，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.