Abstract
Subsaturating amounts of Bacillus subtilis SsbA, independently of the order of addition, partially inhibit the single-stranded DNA-dependent dATPase activity of RecA. This negative effect is fully overcome when a substoichiometric amount of RecO is added. SsbA added prior to RecA does not stimulate the dATP-dependent DNA strand exchange activity; however, added after RecA it enhances the extent of strand exchange. The addition of RecO stimulates RecA-mediated joint molecule formation, although it limits the accumulation of final recombination products. Thus we suggest that RecO has a dual activity: RecO acts as a RecA mediator enabling RecA to utilize SsbA-coated single-stranded DNA as a polymerization substrate and controls RecA-mediated DNA strand exchange by limiting its extent. We herein discuss the possible mechanisms of RecO involvement in the regulation of double strand break repair and genetic transformation.
Highlights
E. coli BL21(DE3) cells bearing the pCB669-borne recO gene were grown to saturation with spontaneous autoinduction of the 29.3-kDa RecO protein, harvested, and resuspended in buffer A containing 300 mM NaCl
Effect of SsbA or SsbSPP1 on RecA Catalyzed ssDNA-dependent dATP Hydrolysis— dATP hydrolysis was used as an indirect measurement of RecA1⁄7dATP1⁄7Mg2ϩ binding onto SsbA- or heterologous SsbSPP1-coated ssDNA
Subsaturating RecA concentrations were used in our experiments to study the effect of SsbA or SsbSPP1 addition in RecA-promoted dATP
Summary
Bacterial Strains and Plasmids—B. subtilis YB886 and E. coli BL21(DE3) (pLysS) and XL1-Blue strains were described previously [39]. pCB596-borne ssbSPP1 [40], pCB722-borne ssbA [5], or pCB669-borne recO genes, under the control of a phage T7 promoter, were used to overexpress SsbSPP1, SsbA, or RecO proteins, respectively. E. coli BL21(DE3) (pLysS) cells bearing the pCB669-borne recO gene were grown to saturation with spontaneous autoinduction of the 29.3-kDa RecO protein, harvested, and resuspended in buffer A containing 300 mM NaCl. The cells were disrupted by the addition of lysozyme (500 ng/ml), followed by sonication. DNA Manipulations—The 3,199-bp KpnI-cleaved dsDNA (20 M in nt) and homologous circular 3,199-nt ssDNA (10 M in nt) were incubated with the indicated concentrations of the indicated protein or a combination of them in buffer C (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 40 mM NaCl, 10 mM magnesium acetate, 50 g/ml bovine serum albumin, 5% glycerol) containing 2 mM dATP for a variable period or for 60 min at 37 °C in a final volume of 20 l. Background scattering due to cuvette, buffer, and ssDNA has been subtracted
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