PcrA-mediated disruption of RecA nucleoprotein filaments--essential role of the ATPase activity of RecA

Abstract

The essential DNA helicase, PcrA, regulates recombination by displacing the recombinase RecA from the DNA. The nucleotide-bound state of RecA determines the stability of its nucleoprotein filaments. Using single-molecule fluorescence approaches, we demonstrate that RecA displacement by a translocating PcrA requires the ATPase activity of the recombinase. We also show that in a 'head-on collision' between a polymerizing RecA filament and a translocating PcrA, the RecA K72R ATPase mutant, but not wild-type RecA, arrests helicase translocation. Our findings demonstrate that translocation of PcrA is not sufficient to displace RecA from the DNA and assigns an essential role for the ATPase activity of RecA in helicase-mediated disruption of its filaments.

Figure 1.

Translocating PcrA disrupts preformed RecA filaments that hydrolyze ATP. (A) Model showing smFRET assay for ATP hydrolysis-dependent RecA displacement from ssDNA by PcrA. The DNA substrate is a tailed duplex labeled with Cy3 (donor) at the 5′-end of the ssDNA tail and Cy5 (acceptor) at the ss/ds junction. Blue, gray and red spheres indicate PcrA, RecA-ATP and RecA-ADP, respectively. RecA filament formation holds the ssDNA tail of the substrate in a stretched state and decreases its FRET. RecA displacement by PcrA leads to an increase in FRET. Repetitive looping of ssDNA by PcrA that remains bound to the ss/ds junction prevents the reassembly of RecA filaments on ssDNA and leads to alternating mid- (free ssDNA) and high-FRET (looped ssDNA) states. (B) FRET histograms of the smFRET DNA substrate (black; indicated as a line for clarity) in the presence of RecA-ATP before (blue) and after (green) the addition of 20 nM PcrA, 1 μM RecA and 1 mM ATP. (C) ATP concentration-dependent repetitive looping of ssDNA by PcrA. Representative graphs of fluorescence intensities of Cy3 (green) and Cy5 (red) and corresponding FRET values (blue) obtained for the smFRET substrate depicted in the presence of 1 nM PcrA and different concentrations of ATP as indicated. (D) Representative graphs of the fluorescence intensities of Cy3 (green) and Cy5 (red) and the corresponding FRET values (blue) obtained for RecA displacement by PcrA in the presence of 1 nM PcrA and 1 mM ATP.

Figure 2.

Selective inhibition of the ATPase activity of RecA prevents disruption of RecA filaments by a translocating PcrA. (A) FRET histograms of the smFRET substrate before and after the addition of 1 μM RecA, 1 mM ATPγS, 1 mM ATP and 10 nM PcrA. FRET histograms for each reaction condition that are closely overlapping are shown in different colors as line graphs. (B) FRET histograms of DNA incubated with 1 mM ATPγS before washing with a buffer containing 1 mM ATP and subsequently after the addition of 10 nM PcrA, 1 μM RecA and 1 mM ATP are shown in different colors.

Figure 3.

Nucleoprotein filaments formed by RecA K72R are disrupted by PcrA with a reduced efficiency compared with wild-type. (A) FRET histograms showing PcrA concentration-dependent displacement of 1 μM RecA and 1 μM K72R, respectively. (B) Comparison of the efficiency with which PcrA disrupts K72R (unshaded) and wild-type RecA (gray) filaments. Data from FRET≤ 0.43 from histograms in (A) were used to calculate clearing efficiency. (C) Comparison of repetitive looping efficiencies of PcrA in the presence of K72R (black) or RecA (gray). Data from FRET ≥ 0.61 from histograms in (A) were used to calculate the looping efficiency.

Figure 4.

Head-on collision between PcrA and RecA arrests translocation of PcrA in the absence of the ATPase activity of RecA. (A) Fluorescence intensities of Cy3 and Cy5 during repetitive looping of ssDNA by 10 nM PcrA on the smFRET substrate before and after the addition of RecA at t = 20 s. Only data up to t = 70 s are shown, although repetitive looping continued for up to 180 s (not shown). Corresponding FRET values are also shown. (B) FRET histogram for experiment shown in (A) before (green histogram) and after (black histogram) the addition of 1 μM RecA at t = 20 s. (C) Fluorescence intensities of Cy3 and Cy5 during an experiment similar to (A) except that 1 μM K72R was added at t = 30 s (black arrow). Corresponding FRET values are also shown. (D) FRET histograms before (green histogram) and after (black histogram) the addition of K72R at t = 30 s for the experiment in (C) showing filament formation by K72R in the presence of translocating PcrA.

Figure 5.

PcrA disrupts low-affinity RecA-ADP filaments but not high-affinity RecA-ADP-AlF₄ filaments. (A) FRET histogram of the smFRET substrate in the presence of 1 μM RecA and 5 mM ADP (blue histogram) and after the addition of 10 nM PcrA, 1 μM RecA and 1 mM ATP (green histogram). (B) FRET histograms of the smFRET substrate after the addition of 1 μM RecA, 5 mM ADP-AlF₄, 1 mM ATP and 10 nM PcrA. For clarity, histograms of closely overlapping populations are represented as line graphs.

Figure 6.

Model for PcrA-mediated disruption of RecA nucleoprotein filaments. Disruption of RecA filaments by a translocating PcrA (blue sphere) requires hydrolysis of ATP by RecA. Red arrow, 3′ to 5′ translocation of PcrA; green arrow, 5′ to 3′ polymerization of RecA. Formation of RecA-ADP, which has lower-affinity for DNA (indicated by the color change of RecA spheres from gray to red) allows PcrA to displace RecA from the DNA and disrupt an entire filament. A monomer of RecA displaced by translocating PcrA is shown as an orange sphere.