Journal:

Article Title: RecR-mediated Modulation of RecF Dimer Specificity for Single- and Double-stranded DNA * S⃞

doi: 10.1074/jbc.M806378200

Figure Lengend Snippet: FRET measurements of ATP-dependent RecF dimerization. A, reactions were carried out as described under “Experimental Procedures.” Signals from FAM and Cy3 labels were measured upon titration of an equimolar mixture of FAM- and Cy3-labeled RecF proteins with ATP or ADP. Changes of FRET signal in arbitrary units were plotted against corresponding nucleotide concentrations. The lanes represent theoretical fit performed with Scientist software and plotted with SigmaPlot 8.0 (SPSS Inc.). Solid, long-dash, medium-dash, short-dash, dotted, and dash-dotted lines with filled symbols correspond to 0.1, 0.2, 0.4, 1, 2, and 4 μm concentrations of RecF, respectively. The solid line with open circles corresponds to titration of 2 μm RecF with ADP. B, a secondary plot of the Bmax values versus the RecF concentrations derived from the FRET measurements shown in A. The curve represents theoretical fitting of data with SigmaPlot.

Article Snippet: The fluorescence polarization signal was plotted against the RecR concentration for each RecF concentration, then analyzed with SigmaPlot 8.0 (SPSS, Inc.) using a single-site ligand-interaction model and Equation 7 , (Eq.

Techniques: Titration, Labeling, Software, Derivative Assay

Journal:

Article Title: RecR-mediated Modulation of RecF Dimer Specificity for Single- and Double-stranded DNA * S⃞

doi: 10.1074/jbc.M806378200

Figure Lengend Snippet: RecF-DNA interaction measured by the fluorescence polarization assay. The labeled DNA substrates (20 nm) were titrated with RecF in the presence of 2 mm nucleotide. RecR was included where indicated at final concentration of 30 μm. Normalized fluorescence polarization signal in arbitrary units was plotted against protein concentrations. The graphs were created with SigmaPlot 8.0 (SPSS Inc.). RecF interactions with ssDNA (S1) are shown in panel A, with dsDNA (D1) in panel B, and with ds/ssDNA (J1) junction in panel C. Dotted lines represent the interaction of the RecF(S279R) mutant with DNA in the presence of ATP; short-dashed lines, a wild-type RecF in the presence of ADP; solid lines, a wild-type RecF in the presence of ATP; long-dashed lines, a wild-type RecF in the presence of ATP and RecR.

Article Snippet: The fluorescence polarization signal was plotted against the RecR concentration for each RecF concentration, then analyzed with SigmaPlot 8.0 (SPSS, Inc.) using a single-site ligand-interaction model and Equation 7 , (Eq.

Techniques: Fluorescence, Labeling, Concentration Assay, Mutagenesis