Rolfsmeier Michael L, Haseltine Cynthia A
School of Molecular Biosciences, Washington State University, Pullman, WA, United States.
School of Molecular Biosciences, Washington State University, Pullman, WA, United States.
Methods Enzymol. 2018;600:255-284. doi: 10.1016/bs.mie.2017.12.009. Epub 2018 Feb 3.
Repair of DNA double-strand breaks is a critical function shared by organisms in all three domains of life. The majority of mechanistic understanding of this process has come from characterization of bacterial and eukaryotic proteins, while significantly less is known about analogous activities in the third, archaeal domain. Despite the physical resemblance of archaea to bacteria, archaeal proteins involved in break repair are remarkably similar to those used by eukaryotes. Investigating the function of the archaeal version of these proteins is, in many cases, simpler than working with eukaryotic homologs owing to their robust nature and ease of purification. In this chapter, we describe methods for purification and activity analysis for the RadA recombinase and its paralogs from the hyperthermophilic acidophilic archaeon Sulfolobus solfataricus.
DNA双链断裂修复是生命所有三个域中的生物体共有的关键功能。对这一过程的大多数机制理解来自对细菌和真核生物蛋白质的表征,而对于第三个域古细菌中的类似活性了解得要少得多。尽管古细菌在形态上与细菌相似,但参与断裂修复的古细菌蛋白质与真核生物使用的蛋白质非常相似。在许多情况下,研究这些蛋白质的古细菌版本的功能比研究真核生物同源物更简单,因为它们性质稳定且易于纯化。在本章中,我们描述了从嗜热嗜酸古细菌嗜热栖热菌中纯化RadA重组酶及其旁系同源物并进行活性分析的方法。