Figure 2
From: ATPase activity tightly regulates RecA nucleofilaments to promote homologous recombination
ATP hydrolysis inhibits the formation of the RecA-saturated nucleofilaments and increases the dynamics from nucleation to extension. (a) CE-LIF electropherograms of the reaction mixtures of TMR-T18 (10 nM) and RecA protein (16.0 μM), showing the formation of six distinct RecA-TMR-T18 complexes induced by ATP hydrolysis. The Y axis of the bottom trace can be applied to all stacked traces in the panel. (b) CE-LIFP electropherograms of the reaction mixtures of TMR-ss83mer (10 nM) and RecA (3.0 μM), showing the formation of three types of RecA nucleofilaments in the presence of ATP. Peaks 1–3 represent the lightly assembled, moderately assembled and RecA-saturated nucleofilaments, respectively. The Y axis of the bottom trace can be applied to all stacked traces in the panel. (c) The relative abundance of the three types of RecA-TMR-ss83mer filaments is dependent on ATP concentration. (d) Denaturing PAGE analysis of time-dependent products of supernuclease digestion of the Cy5-ss83mer (20 nM) and the reaction mixtures of Cy5-ss83mer (20 nM), RecA (3.0 μM) and one nucleotide cofactor as indicated. M indicates ssDNA size markers with a unit of nucleotides (nts). The reactions proceeded at 37 °C for 10 min.
