Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, United States.
Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health & Medical Research Institute, Wollongong, NSW, Australia.
Methods Enzymol. 2022;673:191-225. doi: 10.1016/bs.mie.2022.03.012. Epub 2022 Apr 5.
R-loop proteins present a stable and robust blockade to the progression of a DNA replication fork during S-phase. The consequences of this block can include mutagenesis and other irreversible chromosomal catastrophes, causing genomic instability and disease. As such, further investigation into the molecular mechanisms underlying R-loop protein resolution is warranted. The critical role of non-replicative accessory helicases in R-loop protein resolution has increasingly come into light in recent years. Such helicases include the Pif1-family, monomeric helicases that have been studied in many different contexts and that have been ascribed to a multitude of separable protective functions in the cell. In this chapter, we present protocols to study R-loop protein resolution by Pif1 helicase at stalled replication forks using purified proteins, both at the biochemical and single-molecule level. Our system uses recombinant proteins expressed in Saccharomyces cerevisiae but could apply to practically any organism of interest due to the high interspecies homology of the proteins involved in DNA replication. The methods we outline are extensible to many systems and should be applicable to studying R-loop clearance by any Superfamily (SF) 1B helicase. These techniques will further enable mechanistic research on these critical but understudied components of the genomic maintenance program.
R 环蛋白在 S 期时会对 DNA 复制叉的前进形成稳定且强大的阻碍。这种阻碍的后果可能包括突变和其他不可逆的染色体灾难，从而导致基因组不稳定和疾病。因此，有必要进一步研究 R 环蛋白解析的分子机制。近年来，非复制辅助解旋酶在 R 环蛋白解析中的关键作用越来越受到关注。这类解旋酶包括 Pif1 家族，这是一种单体解旋酶，在许多不同的环境中都有研究，并被赋予了细胞中多种可分离的保护功能。在本章中，我们提供了使用纯化蛋白在停滞的复制叉上通过 Pif1 解旋酶研究 R 环蛋白解析的方案，包括生化和单分子水平。我们的系统使用在酿酒酵母中表达的重组蛋白，但由于涉及 DNA 复制的蛋白质在物种间具有高度同源性，因此几乎可以应用于任何感兴趣的生物体。我们概述的方法可扩展到许多系统，并且应该适用于研究任何 SF1B 解旋酶的 R 环清除。这些技术将进一步推动对基因组维护计划中这些关键但研究不足的成分的机制研究。
