Figure 1.
The binding of Herbaspirillum seropedicae RecA to RecX. The interaction between the H. seropedicae RecA and RecX proteins was determined by a Sepharose-Ni2+ bead immobilization assay as described in Methods. The HisTagged protein immobilized on Ni2+ beads was incubated with the native protein and then eluted with 0.5 M imidazole. A, Lane M = molecular weight markers; lane 1 = native RecA (4.2 μg) and RecXHis (4.2 μg) control; lane 2 = fraction eluted with 0.5 M imidazole from RecXHis-charged Ni2+ beads after the addition of native RecA. B, Lane 1 = DraGHis (4.2 μg) and native RecA (4.2 μg); lane 2 = washing fraction from DraGHis-charged Ni2+ beads; lane 3 = washing fraction of the system DraGHis-Ni2+ beads after the addition of native RecA; lane 4 = fraction eluted with 0.5 M imidazole from DraGHis-charged Ni2+ beads after the addition of native RecA; lane 5 = native RecA (4.2 μg); lane 6 = fraction eluted with 0.5 M imidazole from native RecA-charged Ni2+ beads. The histidine-tagged proteins were immobilized on Sepharose-chelating FF beads (Ni2+ beads, 15 µL) and washed with buffer A (10 mM Tris-HCl, pH 8.0, 50 mM NaCl and 1 mM DTT) containing 5% glycerol in the presence or absence of 2.5 µM (8.4 µg) of native RecA as indicated. The immobilized proteins were eluted with buffer A containing 5% glycerol and 0.5 M imidazole.
Figure 2.
Inhibition by RecX of RecA-mediated DNA strand exchange and ATP binding. Effect of increasing concentrations of the RecXHs protein on the DNA strand exchange mediated by Herbaspirillum seropedicae RecA (RecAHs) (A) or Escherichia coli RecA (RecAEc) (B). Effect of RecXHs on the RecA ATP-binding activity (C). (+) indicates the presence and (-) the absence of the indicated components. S1 is circular ssDNA, S2 is linear dsDNA and P is nicked circular dsDNA.
Figure 3.
Effect of the RecXHs protein concentration (A), the order of its addition (B) and the effect of ssDNA (C) on the ATP hydrolysis mediated by RecAHs. A, The RecAHs protein, fX174 circular ssDNA and increasing concentrations of RecXHs protein (0 µM, filled triangles; 0.1 µm, filled squares; 0.5 µm, open squares; 2 µm, crosses; 5 µm, open triangles; 10 µM, filled circles) were incubated in ATPase buffer at 37°C for 10 min. After this period, the SSB protein and [α32P]-ATP were added. As a control without RecA, 5 µM RecXHs was incubated with ssDNA (open lozenges). B, Two of the following components: fX174 circular ssDNA, RecAHs and RecXHs proteins were pre-incubated in ATPase buffer at 37°C for 10 min. After this period, the third component (RecX, ssDNA or RecA) was added together with the SSB protein and [α32P]-ATP (filled squares, filled circles, open squares). Alternatively, all three components were pre-incubated together under the same conditions, before the addition of the SSB protein and [α32P]-ATP after 10 min (crosses). As controls, RecAHs was incubated with ssDNA in the absence of RecX (filled triangles) or incubated with RecXHs in the absence of ssDNA (open lozenges). C, RecXHs (0.5 or 5 µM) and fX174 circular ssDNA were pre-incubated in ATPase buffer at 37°C for 10 min. After this period, RecA alone (filled lozenges, open triangles), RecA plus ssDNA (132 µM, open squares) or RecA and then ssDNA (132 µM, added just 20 min later, as indicated by the gray arrow; filled squares) were added together with the SSB protein and [α32P]-ATP. The concentration of RecAHs was unchanged and ssDNA[↑] and RecX[↓] indicate the following unusual concentrations: 132 µM ssDNA and 0.5 µM RecX, respectively. In all of the assays after the addition of [α32P]-ATP (zero time), aliquots were collected and stopped.