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. 2018;600:307-320.
doi: 10.1016/bs.mie.2017.12.003. Epub 2018 Jan 9.

Purification of Saccharomyces Cerevisiae Homologous Recombination Proteins Dmc1 and Rdh54/Tid1 and a Fluorescent D-Loop Assay

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Free PMC article

Purification of Saccharomyces Cerevisiae Homologous Recombination Proteins Dmc1 and Rdh54/Tid1 and a Fluorescent D-Loop Assay

Yuen-Ling Chan et al. Methods Enzymol. .
Free PMC article

Abstract

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.

Keywords: Dmc1; Fluorescent D-loop assay; Overexpression; Purification; Rdh54; Saccharomyces cerevisiae; Strand exchange; Tid1.

Figures

Fig. 1
Fig. 1. Comparison of D-loop assays using Cy5-ssDNA vs 32P-ssDNA.
(A) Cartoon of the D-loop assay. (B) Hop2-Mnd1 (H) stimulation of Dmc1 (D) D-loop formation using both fluorescent Cy5-ssDNA and radioactive 32P-ssDNA in the same reaction mixture. Equal amount (15 nM each) of the two ssDNAs were added to the reaction. The “0” lane has no protein. After the D-loop formation step, the reaction mixture was diluted by half, 5 fold, and 10 fold using reaction buffer. The dilution step was followed by deproteinzation and other steps. After gel electrophoresis, the agarose gel was first imaged directly for Cy5 signal in Typhoon 9200 Imager. Subsequently, the same gel was dried onto a positively charged membrane, and exposed to an image plate overnight. The radioactive signal from the image plate was acquired using the same Imager. (C) The signal of D-loop bands from diluted reactions in (B) were quantified and plotted. (D) The D-loop activity of the reactions are plotted.
Fig. 2
Fig. 2
(A) A flow chart showing steps of the two-column method in the purification of (His)6-tagged Dmc1 and Rdh54/Tid1. (B) Overexpression and purification of Dmc1 and Rdh54/Tid1 proteins. Lysates from Rosetta(DE3)pLysS E. coli cells before and after induction of expression by IPTG were analyzed on 12% SDS-PAGE for Dmc1 and 8% for Rdh/Tid1. Gel staining was with Coomassie Brilliant Blue. The purified proteins (2 μg Dmc1 [37 kDa], 2.5 μg Rdh54/Tid1 [105 KDa]) were analyzed on separate gels. Arrows indicate the position of Dmc1 and Rdh54/Tid1; an asterisk indicates the position of the 75 kDa C-terminal Rdh54/Tid1 fragment (4 μg fragment). The intact Rdh54/Tid1 was separated from the truncated protein by Mono S column.

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