Figure 2.

RPA is needed for Rad52 protein-mediated annealing of heat-denatured plasmid DNA. (A) A schematic illustration for the annealing of linear heat-denatured plasmid DNA. (B) RPA (65 nM) and Rad52 protein (30 nM) were added to linear heat-denatured pBluescriptSK+ dsDNA (800 nM) in the indicated order, and annealing was monitored fluorimetrically. Two curves, Rad52 and Rad52 → RPA, are overlaid. The contribution of DNA secondary structure was subtracted. No significant spontaneous annealing occurred in the absence of protein (data not shown). The effect of RPA concentration (C) was examined under the same conditions used in B, and the effect of Rad52 protein concentration (D) was examined under the same conditions, except that RPA concentration was 30 nM. Relative annealing initial rates were determined from the slopes of the fluorescence–time curves.