Figure 6
From: ATPase activity tightly regulates RecA nucleofilaments to promote homologous recombination
RecA mutations at P67 favoring the RecA-saturated nucleofilaments fail to mediate HR in vivo and those favoring the lightly assembled nucleofilaments can efficiently mediate HR in vivo. (a) ATP hydrolytic activity of five P67-mutated RecA proteins. (b) CE-LIFP electropherograms of the P67-mutated RecA-ssDNA filaments when stimulated by physiologically relevant ATP concentration (1.0 mM). The reactions also contained 10.0 nM TMR-ss83mer, 3.0 μM P67-mutated RecA protein as indicated in the figure, and proceeded at 37 °C for 10 min. Peaks 1–3 represent the lightly assembled, moderately assembled and saturated nucleofilaments, respectively. IS indicates the internal migration marker. (c, d) FACS analysis of E. coli HR efficiency of five RecA P67-mutants (c) and the percentage of GFP+ cells (d). FITC-H and PE-H (c) indicate the GFP signal and background fluorescence of the sorted E. coli cells, respectively. Pink and blue dots indicate GFP+ and GFP− cells, respectively.
