Purification of Saccharomyces cerevisiae Homologous Recombination Proteins Dmc1 and Rdh54/Tid1 and a Fluorescent D-Loop Assay
| Autor: | Yuen-Ling Chan, Douglas K. Bishop |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Saccharomyces cerevisiae Proteins Saccharomyces cerevisiae DNA Single-Stranded Cell Cycle Proteins Article Homology (biology) law.invention 03 medical and health sciences chemistry.chemical_compound D-loop Meiosis law Chromatography High Pressure Liquid Fluorescent Dyes 030102 biochemistry & molecular biology biology fungi DNA Helicases Recombinational DNA Repair Chromatography Ion Exchange biology.organism_classification Recombinant Proteins DNA-Binding Proteins 030104 developmental biology Biochemistry chemistry Recombinant DNA DMC1 Homologous recombination DNA Topoisomerases DNA |
| DOI: | 10.1016/bs.mie.2017.12.003 |
| Popis: | Budding yeast Dmc1 is a member of the RecA family of strand exchange proteins essential for homologous recombination (HR) during meiosis. Dmc1 mediates the steps of homology search and DNA strand exchange reactions that are central to HR. To achieve optimum activity, Dmc1 requires a number of accessory factors. Although methods for purification of Dmc1 and many of its associated factors have been described (Binz, Dickson, Haring, & Wold, 2006; Busygina et al., 2013; Chan, Brown, Qin, Handa, & Bishop, 2014; Chi et al., 2006; Cloud, Chan, Grubb, Budke, & Bishop, 2012; Nimonkar, Amitani, Baskin, & Kowalczykowski, 2007; Van Komen, Macris, Sehorn, & Sung, 2006), Dmc1 has been particularly difficult to purify because of its tendency to aggregate. Here, we provide an alternative and simple high-yield purification method for recombinant Dmc1 that is active and responsive to stimulation by accessory factors. The same method may be used for purification of recombinant Rdh54 (a.k.a. Tid1) and other HR proteins with minor adjustments. We also describe an economical and sensitive D-loop assay for strand exchange proteins that uses fluorescent dye-tagged, rather than radioactive, ssDNA substrates. |
| Databáze: | OpenAIRE |
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