<i>B</i>. <i>burgdorferi</i> RecA-promoted strand exchange with oligonucleotide substrates is strongly stimulated by SSB.

<p>A) Schematic representation of the oligonucleotide substrates used. The ssDNA donor oligonucleotide is 70 nt in length while the 5’-endlabeled duplex target DNA is 35 bp. * denote the ³²P-endlabels, red/shaded lines represent endlabeled strands.B) 8% PAGE 1X TAE/0.1% SDS gel analysis of strand exchange reactions with RecA, -/+ SSB. Strand exchange was assayed in a buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl₂, 1 mM DTT, 100 μg/ml BSA, 50 mM NaCl and 2 mM ATP, AMPPNP or ATP[γS] in 60 μL reaction volumes. The assay was conducted as a staged reaction with pre-incubation of RecA with the donor ssDNA (30°C, 5 min) followed by addition of 5’-endlabeled duplex target DNA (S) and continued incubation at 37°C. RecA was present at 2 μM, donor ssDNA at 1.4 μM (nt) and 5’-endlabeled target duplex (S) was present at 1.4 μM (nt). When added, SSB was added after pre-incubation of RecA with the single stranded donor and was present at 300 nM. The proteins and nucleotide co-factor present are indicated in the loading key above the gels. C) 8% PAGE 1X TAE/0.1% SDS gel analysis of strand exchange reactions with RecA and a titration of SSB added before RecA (1st) or after RecA incubation with single-stranded donor DNA (2nd). Reactions were performed with ATP[γS] as the nucleotide co-factor.</p

<i>B</i>. <i>burgdorferi</i> RecA binds preferentially to ssDNA.

<p>6% PAGE 0.5X TBE gel analysis of RecA binding to 95 nt ssDNA and 95 bp dsDNA. The indicated concentrations of RecA were assayed for binding with 10 nM (molecules) of the indicated 5-endlabeled DNAs in a buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl₂, 1 mM DTT, 50 mM NaCl and, where indicated, 2 mM ATP, by incubation at 30°C for 5 min. This was followed by protein crosslinking with glutaraldehyde (final concentration 0.033%) at 20°C for 5 min prior to application of the binding reactions to native a PAGE gel. The gel migration position of RecA-DNA complexes is indicated by asterisks and the position of the wells is noted. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187382#sec002" target="_blank">Materials and methods</a> section for details.</p

<i>B</i>. <i>burgdorferi</i> RecA binds ATP and possesses DNA-dependent ATPase activity.

<p>A) 12% SDS-PAGE analysis of RecA ATP photoaffinity binding assays. ATP binding was assayed by incubating 2 μM RecA in reaction buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl₂, 1 mM DTT, 50 mM NaCl, 25 μM ATP and 66 nM [γ³²P]ATP for 5 min on ice followed by photocrosslinking (312 nm) for the times indicated above the gel. Where indicated, ϕX174 virion DNA was present at 10 μg/ml. M denotes the protein molecular weight marker. B) Polyethyleneimine thin-layer chromatography ATPase assay results with the indicated concentrations of RecA -/+ 10 μg/mL ϕX174 virion (ssDNA) and ϕX174 RF1 (dsDNA). ATPase activity was assayed by incubating the indicated concentrations of RecA in buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl₂, 1 mM DTT, 100 μg/ml bovine serum, 50 mM NaCl, 100 μM ATP and 66 nM [γ³²P]ATP in a total volume of 30 μL for 2 hours. M denotes a 2 hour mock incubation of [γ³²P]ATP without added DNA or RecA. C) Summary of ATPase assay results with <i>B</i>. <i>burgdorferi vs</i>. <i>E</i>. <i>coli</i> RecA, -/+ 10 μg/mL ϕX174 virion or ϕX174 RF1 DNA. ATPase assays containing 1 μM RecA were incubated at 37°C for 5 min for ϕX174 virion (ssDNA) containing reactions or at 37°C for 120 min for DNA-free and ϕX174 RF1 (dsDNA) containing reactions. The mean and standard deviation of at least three independent experiments is shown.</p

Biochemical characterization of <i>Borrelia burgdorferi</i>’s RecA protein

<div><p>RecA plays key roles in DNA recombination, replication and repair. Mutation of <i>recA</i> in the Lyme disease spirochete, <i>Borrelia burgdorferi</i>, fails to produce some of the phenotypes expected from study of <i>recA</i> mutation in other organisms. ‘Missing’ <i>recA</i> phenotypes include a lack of growth or viability effects, including in the presence of DNA damage, and a lack of a role in <i>vlsE</i> antigenic variation and infectivity. We present a purification and biochemical characterization of recombinant <i>B</i>. <i>burgdorferi</i> RecA protein. We find that <i>B</i>. <i>burgdorferi</i> RecA displays the expected properties of being a DNA-dependent ATPase, of having an intrinsic binding preference for ssDNA over dsDNA enhanced by ATP binding, of promoting DNA pairing and strand exchange reactions and of having a detectable coprotease activity with <i>E</i>. <i>coli</i> LexA repressor. DNA pairing and strand exchange reactions promoted by <i>B</i>. <i>burgdorferi</i> RecA show an unusually strong dependence upon the presence of the cognate ssDNA binding protein (SSB) but are very sensitive to inhibition by SSB when the ssDNA was prebound by SSB. This indicates <i>B</i>. <i>burgdorferi</i> RecA may have an enhanced requirement for recombinational mediators to promote RecA-SSB exchange, despite the absence of homologues of the RecF pathway proteins that normally play this role in eubacteria. Finally, we do not find any unusual, intrinsic properties of <i>B</i>. <i>burgdorferi</i>’s RecA protein to explain the unusual phenotype of <i>recA</i> mutation and suggest that there may be alternative recombinase functions that could explain the ‘missing’ phenotypes.</p></div

<i>B</i>. <i>burgdorferi</i> RecA possesses a weak DNA-independent co-protease activity with <i>E</i>. <i>coli</i> LexA.

<p>A) 5/18% SDS-PAGE analysis of nucleotide and DNA requirements for co-protease activity with <i>E</i>. <i>coli</i> LexA. Reactions were performed as detailed in the Materials and methods by incubation of RecA/LexA or LexA with the buffer components indicated in the gel-loading key at 30°C for 16 h. RecA was present at 1.25 μM, LexA at 1.5 μM, and where indicated, donor ssDNA was present in excess of RecA (2 μM nt). The gel was visualized by Coomassie Brilliant Blue staining. M denotes loading controls for each protein to mark their gel-migration position, CL denotes the LexA cleavage fragments. The asterisk in somes lanes of the LexA only incubation controls corresponds to the migration position of a LexA dimer. We infer that some LexA dimers formed during the overnight incubation (through cysteine oxidation) and were not completely disrupted before gel loading. B) 5/18% SDS-PAGE analysis of the effect of supplementing co-protease reactions with <i>B</i>. <i>burgdorferi</i> SSB. RecA was present at 1.25 μM, LexA at 1.5 μM, SSB at 0.19 μM and where indicated donor ssDNA was present at 0.5 μM (nt). To aid visualization of low abundance SSB the gel was silver stained subsequent to Coomassie Brilliant Blue staining. M denotes loading controls for each protein to mark their gel-migration position, CL denotes the LexA cleavage fragments.</p

Strand invasion is highly reversible when RecA can hydrolyze ATP.

<p>A) Schematic representation of the strand invasion assay indicating the effect of SSB order of addition and ATP hydrolysis on D-loop recovery. B) 0.8% agarose 1X TAE gel analysis of strand invasion timecourses performed -/+ 0.3 μM SSB addition (added 2^nd vs. added 1^st) in buffer containing ATP (top panel) or AMPPNP (bottom panel). Reaction conditions were as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187382#pone.0187382.g003" target="_blank">Fig 3</a> and in the Materials and methods section. C) 0.8% agarose 1X TAE gel analysis of strand invasion timecourses performed -/+ 0.3 μM SSB addition (added 2^nd) comparing the behavior of wild type RecA <i>vs</i>. the Walker A mutant, RecA (K88R) in a buffer containing ATP.</p

<i>B</i>. <i>burgdorferi</i> RecA-promoted strand exchange with long substrates is dependent upon SSB.

<p>0.8% agarose 1X TAE gel analysis of strand exchange reactions performed with RecA and a titration of SSB in a buffer with hydrolysable ATP. RecA was present at 2 μM, ϕX174 virion (css) at 5.1 μM (nt) and XhoI-linearized ϕX174 duplex DNA (lds) at 5.1 μM (nt). When present, SSB was added after RecA pre-incubation with ϕX174 virion and was present at the concentration noted in the loading key above the gel. The gel labels are as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187382#pone.0187382.g006" target="_blank">Fig 6</a>.</p

<i>B</i>. <i>burgdorferi</i> RecA-promoted strand exchange with long substrates.

<p>A) Schematic of the strand exchange assay using ϕX174 virion DNA (closed circular ssDNA; css) and XhoI-linearized ϕX174 duplex DNA (linear dsDNA; lds). As strand exchange initiates, joint molecule (JM) intermediates form. As strand exchange progresses, the final strand exchange products of nicked circular DNA (NC) and linear ssDNA (lss) are formed. Partial, intermolecular strand exchange between multiple donor and target molecules can also give rise to large aggregates (aggr.). B) 0.8% agarose 1X TAE gel analysis of strand exchange reactions performed with RecA and SSB. Strand exchange was assayed in a buffer containing 25 mM HEPES (pH 7.6), 1 mM DTT, 100 μg/ml BSA, 50 mM NaCl, 2 mM ATP and the indicated concentration of MgCl₂ in 120 μL reaction volumes. The assay was conducted as a staged reaction with pre-incubation of RecA with the donor ssDNA (css; 30°C, 5 min) followed by addition of SSB and linear duplex DNA (lds) and continued incubation at 37°C. RecA was present at 2 μM, ϕX174 virion (css) at 5.1 μM (nt) and XhoI-linearized ϕX174 duplex DNA (lds) at 5.1 μM (nt). SSB was added after pre-incubation of RecA with the single stranded donor and was present at 0.9 μM. The concentration of added MgCl₂ is noted in the loading key above the gel. The migration position of the substrate DNA is noted to the left of the gel and of the products to the right. Under our gel conditions css and lss have identical gel mobilities. M denotes mock incubation of the substrate DNAs without added RecA or SSB. C) 0.8% agarose 1X TAE gel analysis of SSB order-of-addition effects and ATP hydrolysis requirements of strand exchange reactions performed with long substrates. Strand exchange reactions were performed as noted in B) using buffer conditions with the optimal 5 mM MgCl₂ concentration and 0.9 μM of added SSB. The order of SSB addition and nucleotide cofactor present is indicated in the loading key above the gel. When added first, SSB was incubated with ϕX174 virion at 30°C for 5 min prior to the addition of RecA and linear ϕX174 duplex DNA followed by continued incubation at 37°C for the times indicated above the gel. When SSB was added second, RecA was incubated with ϕX174 virion at 30°C for 5 min prior to the addition of SSB and linear duplex DNA, followed by continued incubation at 37°C for the times indicated above the gel. M denotes mock incubation of the substrate DNAs without added RecA or SSB.</p