Fig. 5.

RecN promotes RecA-mediated D-loop formation and DNA strand exchange. a D-loop assays. RecA (2 µM) was incubated for 10 min in the presence of the pUC19-derivative linear ds/ssDNA (probe), and then RecN (1 µM) was added to the reaction mixture, followed by incubation for a further 15 min. The reactions were initiated by adding homologous pUC19 supercoiled circular DNA (target) and then incubated for another 5 min. The samples were analyzed by agarose gel electrophoresis and SYBR Gold staining. b Quantification of the amounts of D-loop products in the gel image shown in a. Data represent the mean ± standard deviation of three independent experiments. c Strand exchange assays. RecA (0.6 µM) was incubated for 10 min in the presence of phiX174 circular ssDNA (Css). The indicated concentrations (0–1.5 µM) of RecN were added to the reaction mixture, followed by incubation for 15 min. Subsequently, ssDNA-binding protein was added and samples were incubated for an additional 10 min. The reactions were initiated by adding homologous phiX174 linear dsDNA (Lds), incubated for 90 min, and then stopped by the addition of stop buffer. The samples were analyzed by agarose gel electrophoresis and SYBR Gold staining. d Quantification of the amounts of nicked circular dsDNA (NC) products in the gel image shown in c. Data represent the mean ± standard deviation of three independent experiments. e Time-course strand exchange experiments. RecA (0.8 µM) was incubated with or without RecN (1 µM). Aliquots were collected at the indicated time points and analyzed as described above. f Quantification of the amounts of NC DNA products in the gel image shown in e. c, e The arrowheads indicate the DNA substrates (Css and Lds) and the resulting products: joint molecules (JM), nicked circular dsDNA (NC), and linear ssDNA (Lss)