Bacillus subtilis RecU Holliday-junction resolvase modulates RecA activities

Abstract

The Bacillus subtilis RecU protein is able to catalyze in vitro DNA strand annealing and Holliday-junction resolution. The interaction between the RecA and RecU proteins, in the presence or absence of a single-stranded binding (SSB) protein, was studied. Substoichiometric amounts of RecU enhanced RecA loading onto single-stranded DNA (ssDNA) and stimulated RecA-catalyzed D-loop formation. However, RecU inhibited the RecA-mediated three-strand exchange reaction and ssDNA-dependent dATP or rATP hydrolysis. The addition of an SSB protein did not reverse the negative effect exerted by RecU on RecA function. Annealing of circular ssDNA and homologous linear 3'-tailed double-stranded DNA by RecU was not affected by the addition of RecA both in the presence and in the absence of SSB. We propose that RecU modulates RecA activities by promoting RecA-catalyzed strand invasion and inhibiting RecA-mediated branch migration, by preventing RecA filament disassembly, and suggest a potential mechanism for the control of resolvasome assembly.

Figure 1

RecU stimulates binding of RecA to ssDNA. A linear 200 nt [γ-³²P]ssDNA (0.75 μM) was pre-incubated with RecA [400 nM (lanes 5–7) or 200 nM (lanes 8–10)] or with RecU [12–48 nM (lanes 15–13 and 18–16)] for 10 min at 37°C in buffer B containing 2 mM dATP. Then, RecU (12–48 nM, lanes 7–5 and 10–8)] or RecA [400 nM (lanes 13–15) or 200 nM (lanes 16–18)] were added and the reaction mixture was incubated for further 10 min at 37°C. As a control, the 200 nt ssDNA was incubated for 20 min at 37°C with increasing concentrations of RecU (12–48 nM, lanes 2–4) or RecA (400 nM in lane 11 and 200 nM in lane 12). Samples were analyzed by 0.8% agarose gel electrophoresis, and autoradiographs of the dried gels were subsequently taken. The order of protein addition is indicated. FD, free ssDNA; PDC, protein–DNA complexes.

Figure 2

Effect of RecU on ssDNA-dependent RecA-catalyzed dATP and rATP hydrolysis. Circular ssDNA (10 μM) was pre-incubated with RecU (12–200 nM) in buffer B containing 0.15 or 0.6 mM dATP or rATP for 10 min at 37°C. Then, RecA (1.3 μM) was added and the reaction mixture incubated for 15 min at 37°C. Reactions were stopped by addition of EDTA, and the rate of dATP or rATP hydrolysis by RecA (denoted in pmoles of dATP or rATP hydrolyzed/min) was measured. Empty squares, RecA dATPase in the presence of 0.6 mM; filled squares, 0.15 mM dATP. Empty circles, RecA rATPase in the presence of 0.6 mM; filled circles, 0.15 mM rATP.

Figure 3

Effect of RecU on ssDNA-dependent RecA catalyzed dATP hydrolysis. Circular ssDNA (10 μM) was pre-incubated with RecA (1.3 μM) [time −10 in (A)] or increasing concentrations of RecU [50, 100 and 200 nM, in (B)] in buffer B containing 2 mM dATP for 10 min at 37°C. Then, the second protein {RecU [50, 100 or 200 nM, in (A)] or RecA [1.3 μM, in (B)], denoted by arrowheads} was added and the reaction mixture incubated for further 60 min at 37°C. In (C and D), the order of protein addition and concentrations are the same as in (A and B), respectively, but circular ssDNA (10 μM) and KpnI-linearized dsDNA (20 μM) were present. Aliquots were taken and the rate of dATP hydrolysis by RecA (denoted in pmoles of dATP hydrolyzed) was measured. RecA alone, circles; RecA and 50 nM RecU, triangles; RecA and 100 nM RecU, squares; RecA and 200 nM RecU, diamonds.

Figure 4

RecU inhibits three-strand exchange catalyzed by RecA. (A) Scheme of the three-strand exchange reaction, with the predicted products of RecU cleavage of the three-strand recombination intermediate, and the expected products after RecA catalyzed partial (jm) or full (nc and displaced lss) strand exchange. (B and C) Circular ssDNA (10 μM) was pre-incubated with a constant amount of RecA (1.3 μM) or increasing concentrations of RecU (50–400 nM) in the presence of homologous KpnI-linearized dsDNA (20 μM) (in B) or 3′-tailed dsDNA (20 μM) (in C) for 30 min at 37°C in buffer B containing 2 mM dATP. When indicated, increasing concentrations of RecU (50–400 nM) or RecA (1.3 μM) were added and the samples incubated for further 30 min at 37°C. (B) In lanes 2–7, RecA was pre-incubated with DNA, and in lanes 9–12 RecA was added after RecU. In lane 2, the reaction was stopped at 30 min and in lanes 3 and 8 at 60 min. (C) In lanes 8–11, RecA was pre-incubated with DNA, and in lanes 12–15 RecU was pre-incubated. In lane 6, the reaction was stopped at 30 min and in lanes 7 and 16 at 60 min. In lanes 2–5, the formation by RecU of jm with 3′-tailed dsDNA is shown. Positions of bands corresponding to circular ssDNA (css), linear, nicked and covalently closed dsDNA (lds, nc and ccc, respectively) and joint molecules (jm) are indicated. The order of protein addition is indicated. + and − denote the presence and absence of the indicated protein. C indicates the partially nicked dsDNA control.

Figure 5

RecU inhibition of RecA-promoted DNA strand exchange is not reversed by SSB. Circular ssDNA (10 μM) was pre-incubated with a constant amount of RecA (1.3 μM, lanes 2–17) in the presence of homologous KpnI-linearized dsDNA (20 μM) (in A) or 3′-tailed dsDNA (20 μM) (in B) for 20 min at 37°C. RecU [50 (lanes 4–7), 100 (lanes 8–12) or 200 nM (lanes 13–17)] was added and the samples were incubated for 10 min at 37°C in buffer B containing 2 mM dATP. Then, increasing concentrations of SSB (270–550 nM, in lanes 6–7 or 130–550 nM, in lanes 10–12 and 15–17) were added and incubation was continued for further 30 min at 37°C. In lane 2, the reaction was stopped at 20 min, and in lanes 4, 8 and 13 after 30 min of incubation. Positions of the bands and the symbols are those of Figure 4.

Figure 6

RecU inhibits DNA strand exchange catalyzed by RecA in the presence of SSB. Circular ssDNA (10 μM) was pre-incubated in buffer B containing 2 mM dATP with a constant amount of RecA (1.3 μM, lanes 6–12) in the presence of homologous KpnI-linearized dsDNA (20 μM) (in A) or 3′-tailed dsDNA (20 μM) (in B) for 20 min at 37°C. SSB (270 nM) was added and after incubation for 10 min at 37°C, increasing concentrations of RecU (50–200 nM) were added and the reaction incubated for further 30 min at 37°C. In lanes 6 and 8, the reaction was stopped at 30 min. In lanes 2–4, the DNA substrates were incubated with RecU alone. Position of the bands and the symbols are those of Figure 4.

Figure 7

Effect of RecU on RecA-promoted strand invasion. A homologous 200 nt [γ³²P]ssDNA (0.12 μM) was pre-incubated with RecA [250 nM, in (A)] or with increasing concentrations of RecU [7.5–250 nM, in (B)] for 10 min at 37°C in buffer B containing 2 mM dATP. Supercoiled homologous DNA (7.5 μM) was added and the reaction incubated for 10 min at 37°C. Then, increasing concentrations of RecU [7.5–250 nM, in (A)] or a fixed amount of RecA [250 nM, in (B)] were added and after 15 min the deproteinized products were analyzed with 0.8% agarose gel electrophoresis; subsequently, autoradiographs of the dried gels were taken. As a control, the 200 nt ssDNA was incubated with supercoiled homologous DNA and 125 (lane 2) or 250 nM (lane 3) RecU or RecA (250 nM, lane 4) alone. The order of protein addition is indicated.