Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
Methods Mol Biol. 2022;2528:305-316. doi: 10.1007/978-1-0716-2477-7_20.
R-loops, three-stranded RNA-DNA hybrids that arise mostly during transcription, could cause genomic instability via distinct routes. Detection of genomic RNA-DNA hybrids via immunofluorescence and RNA-DNA hybrid immunoprecipitation techniques have facilitated the discovery of many cellular factors that maintain R-loop homeostasis. One of multiple R-loop avoidance mechanisms is mediated by several nucleic acid motor proteins that utilize the energy from ATP hydrolysis to dissociate the R-loop structure. The biochemical activity of such motor proteins can be interrogated using synthetic R-loop substrates. Here, we describe methods to generate R-loop and RNA-DNA substrates for studying the activity of R-loop processing motor proteins such as human DHX9 and S. cerevisiae Pif1. Such studies provide valuable information regarding the directionality, nucleic acid strand preference, and processivity of these enzymes. Moreover, these substrates and companion biochemical assays provide the requisite tool for understanding the action of physiologically relevant regulators of these motor proteins.
R 环是在转录过程中主要形成的三链 RNA-DNA 杂交体，可通过不同途径导致基因组不稳定。通过免疫荧光和 RNA-DNA 杂交免疫沉淀技术检测基因组 RNA-DNA 杂交体，促进了许多维持 R 环动态平衡的细胞因子的发现。多种 R 环回避机制之一是由几种核酸马达蛋白介导的，这些蛋白利用 ATP 水解的能量解离 R 环结构。可以使用合成的 R 环底物来检测这些马达蛋白的生化活性。在这里，我们描述了生成 R 环和 RNA-DNA 底物的方法，用于研究 R 环加工马达蛋白的活性，如人类 DHX9 和酿酒酵母 Pif1。这些研究提供了有关这些酶的方向性、核酸链偏好性和连续性的有价值的信息。此外，这些底物和伴随的生化测定为理解这些马达蛋白的生理相关调节剂的作用提供了必要的工具。
