Journal: PLoS ONE
Article Title: Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing
Figure Lengend Snippet: Mediator assay of yRad52 with hRAD51. (A) Illustration of the DNA strand exchange experiment to analyze the mediator activity. ΦX-174 ssDNA was incubated with yRPA, yRad52, hRAD51, and then with ΦX-174 dsDNA in the indicated order. The reaction produced joint molecules (jm) and a nicked circular (nc) dsDNA. (B) Derivatives of yRad52. N-terminal (N), middle (M), and C-terminal (C) region have been described in previous paper [ 18 ]. For bottom three constructs, yRad52NM was fused with a human BRC4 (NM-BRC4), BRC3-BRC4 (NM-BRC3-4), or three repeats of BRC4 (NM-BRC4 x3 ). (C) Same amount (2 μg) of purified yRad52 and its derivatives were separated by SDS-PAGE and stained with coomassie brilliant blue. Asterisk indicates a contaminating protein present in all preparations. (D and E) DNA strand exchange was performed in the absence (lane 2) or presence of 1, 2, 3, 4 μM (lane 3 to 6) of yRad52 (D) or yRad52NM (E). DNA products were separated through agarose gel and visualized with ethidium bromide staining. Lane 1 shows a control reaction without any protein. (F) The products (nicked circles (nc) and joint molecules (jm)) were quantified from D (yRad52) and E (yRad52NM) and repeated experiments and relative product formation was plotted against the mediator concentration. Product formation in the absence of the mediator was 31.2%, which was defined as 1.0. Error bars are standard deviations (n = 3). (G) hRAD51 and His-tagged yRad52 were mixed as indicated and precipitated with Ni-beads. Proteins on the beads were then eluted and analyzed by SDS-PAGE.
Article Snippet: Mediator assay ΦX174 phage circular ssDNA and dsDNA were purchased from New England Biolabs and the dsDNA was linearized using Xho I. RPA and ssDNA were incubated for 2 minutes at 37°C in buffer containing 40 mM Tris-HCl (pH 7.5), 1 mM DTT, 2 mM ATP, 1 mM MgCl2 , 8 mM creatine phosphate, and 28 μg/ml creatine phosphokinase.
Techniques: Activity Assay, Incubation, Produced, Construct, Purification, SDS Page, Staining, Agarose Gel Electrophoresis, Concentration Assay