RecF Pathway Research Articles

Differential requirement for RecFOR pathway components in Thermus thermophilus.

Recombinational repair is an important mechanism that allows DNA replication to overcome damaged templates, so the DNA is duplicated timely and correctly. The RecFOR pathway is one of the common ways to load RecA, while the RuvABC complex operates in the resolution of DNA intermediates. We have generated deletions of recO, recR and ruvB genes in Thermus thermophilus, while a recF null mutant could not be obtained. The recO deletion was in all cases accompanied by spontaneous loss of function mutations in addA or addB genes, which encode a helicase-exonuclease also key for recombination. The mutants were moderately affected in viability and chromosome segregation. When we generated these mutations in a Δppol/addAB strain, we observed that the transformation efficiency was maintained at the typical level of Δppol/addAB, which is 100-fold higher than that of the wild type. Most mutants showed increased filamentation phenotypes, especially ruvB, which also had DNA repair defects. These results suggest that in T. thermophilus (i) the components of the RecFOR pathway have differential roles, (ii) there is an epistatic relationship of the AddAB complex over the RecFOR pathway and (iii) that neither of the two pathways or their combination is strictly required for viability although they are necessary for normal DNA repair and chromosome segregation.

Allosteric effects of E. coli SSB and RecR proteins on RecO protein binding to DNA.

Escherichia coli single stranded (ss) DNA binding protein (SSB) plays essential roles in DNA maintenance. It binds ssDNA with high affinity through its N-terminal DNA binding core and recruits at least 17 different SSB interacting proteins (SIPs) that are involved in DNA replication, recombination, and repair via its nine amino acid acidic tip (SSB-Ct). E. coli RecO, a SIP, is an essential recombination mediator protein in the RecF pathway of DNA repair that binds ssDNA and forms a complex with E. coli RecR protein. Here, we report ssDNA binding studies of RecO and the effects of a 15 amino acid peptide containing the SSB-Ct monitored by light scattering, confocal microscope imaging, and analytical ultracentrifugation (AUC). We find that one RecO monomer can bind the oligodeoxythymidylate, (dT)15, while two RecO monomers can bind (dT)35 in the presence of the SSB-Ct peptide. When RecO is in molar excess over ssDNA, large RecO-ssDNA aggregates occur that form with higher propensity on ssDNA of increasing length. Binding of RecO to the SSB-Ct peptide inhibits RecO-ssDNA aggregation. RecOR complexes can bind ssDNA via RecO, but aggregation is suppressed even in the absence of the SSB-Ct peptide, demonstrating an allosteric effect of RecR on RecO binding to ssDNA. Under conditions where RecO binds ssDNA but does not form aggregates, SSB-Ct binding enhances the affinity of RecO for ssDNA. For RecOR complexes bound to ssDNA, we also observe a shift in RecOR complex equilibrium towards a RecR4O complex upon binding SSB-Ct. These results suggest a mechanism by which SSB recruits RecOR to facilitate loading of RecA onto ssDNA gaps.

NtcA, LexA and heptamer repeats involved in the multifaceted regulation of DNA repair genes recF, recO and recR in the cyanobacterium Nostoc PCC7120

Regulation of DNA repair genes in cyanobacteria is an unexplored field despite some of them exhibiting high radio-resistance. With RecF pathway speculated to be the major double strand break repair pathway in Nostoc sp. strain PCC7120, regulation of recF, recO and recR genes was investigated. Bioinformatic approach-based identification of promoter and regulatory elements was validated using qRT-PCR analysis, reporter gene and DNA binding assays. Different deletion constructs of the upstream regulatory regions of these genes were analysed in host Nostoc as well as heterologous system Escherichia coli. Studies revealed: (1) Positive regulation of all three genes by NtcA, (2) Negative regulation by LexA, (3) Involvement of contiguous heptamer repeats with/without its yet to be identified interacting partner in regulating (i) binding of NtcA and LexA to recO promoter and its translation, (ii) transcription or translation of recF, (4) Translational regulation of recF and recO through non-canonical and distant S.D. sequence and of recR through a rare initiation codon. Presence of NtcA either precludes binding of LexA to AnLexA-Box or negates its repressive action resulting in higher expression of these genes under nitrogen-fixing conditions in Nostoc. Thus, in Nostoc, expression of recF, recO and recR genes is intricately regulated through multiple regulatory elements/proteins. Contiguous heptamer repeats present across the Nostoc genome in the vicinity of start codon or promoter is likely to have a global regulatory role. This is the first report detailing regulation of DSB repair genes in any algae.

A Robust One-Step Recombineering System for Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3–4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates.

Single strand gap repair: The presynaptic phase plays a pivotal role in modulating lesion tolerance pathways.

During replication, the presence of unrepaired lesions results in the formation of single stranded DNA (ssDNA) gaps that need to be repaired to preserve genome integrity and cell survival. All organisms have evolved two major lesion tolerance pathways to continue replication: Translesion Synthesis (TLS), potentially mutagenic, and Homology Directed Gap Repair (HDGR), that relies on homologous recombination. In Escherichia coli, the RecF pathway repairs such ssDNA gaps by processing them to produce a recombinogenic RecA nucleofilament during the presynaptic phase. In this study, we show that the presynaptic phase is crucial for modulating lesion tolerance pathways since the competition between TLS and HDGR occurs at this stage. Impairing either the extension of the ssDNA gap (mediated by the nuclease RecJ and the helicase RecQ) or the loading of RecA (mediated by RecFOR) leads to a decrease in HDGR and a concomitant increase in TLS. Hence, we conclude that defects in the presynaptic phase delay the formation of the D-loop and increase the time window allowed for TLS. In contrast, we show that a defect in the postsynaptic phase that impairs HDGR does not lead to an increase in TLS. Unexpectedly, we also reveal a strong genetic interaction between recF and recJ genes, that results in a recA deficient-like phenotype in which HDGR is almost completely abolished.

The rarA gene as part of an expanded RecFOR recombination pathway: Negative epistasis and synthetic lethality with ruvB, recG, and recQ

The RarA protein, homologous to human WRNIP1 and yeast MgsA, is a AAA+ ATPase and one of the most highly conserved DNA repair proteins. With an apparent role in the repair of stalled or collapsed replication forks, the molecular function of this protein family remains obscure. Here, we demonstrate that RarA acts in late stages of recombinational DNA repair of post-replication gaps. A deletion of most of the rarA gene, when paired with a deletion of ruvB or ruvC, produces a growth defect, a strong synergistic increase in sensitivity to DNA damaging agents, cell elongation, and an increase in SOS induction. Except for SOS induction, these effects are all suppressed by inactivating recF, recO, or recJ, indicating that RarA, along with RuvB, acts downstream of RecA. SOS induction increases dramatically in a rarA ruvB recF/O triple mutant, suggesting the generation of large amounts of unrepaired ssDNA. The rarA ruvB defects are not suppressed (and in fact slightly increased) by recB inactivation, suggesting RarA acts primarily downstream of RecA in post-replication gaps rather than in double strand break repair. Inactivating rarA, ruvB and recG together is synthetically lethal, an outcome again suppressed by inactivation of recF, recO, or recJ. A rarA ruvB recQ triple deletion mutant is also inviable. Together, the results suggest the existence of multiple pathways, perhaps overlapping, for the resolution or reversal of recombination intermediates created by RecA protein in post-replication gaps within the broader RecF pathway. One of these paths involves RarA.

Revival of Anhydrobiotic Cyanobacterium Biofilms Exposed to Space Vacuum and Prolonged Dryness: Implications for Future Missions beyond Low Earth Orbit.

Dried biofilms of Chroococcidiopsis sp. CCMEE 029 were revived after a 672-day exposure to space vacuum outside the International Space Station during the EXPOSE-R2 space mission. After retrieval, they were air-dried stored for 3.5 years. Space vacuum reduced cell viability and increased DNA damage compared to air-dried storage for 6 years under laboratory conditions. Long exposure times to space vacuum and extreme dryness decrease the changes of survival that ultimately depend on DNA damage repair upon rehydration, and hence, an in silico analysis of Chroococcidiopsis sp. CCMEE 029's genome was performed with a focus on DNA repair pathways. The analysis identified a high number of genes that encode proteins of the homologous recombination RecF pathway and base excision repair that were over-expressed during 1 and 6 h rehydration of space-vacuum exposed biofilms. This suggests that Chroococcidiopsis developed a survival strategy against desiccation, with DNA repair playing a key role, which allowed the revival of biofilms exposed to space vacuum. Unravelling how long anhydrobiotic cyanobacteria can persist under space vacuum followed by prolonged air-dried storage is relevant to future astrobiological experiments that use space platforms and might require prolonged air-dried storage of the exposed samples before retrieval to Earth.

Rec(F/O/R) proteins of the nitrogen-fixing cyanobacterium Nostoc PCC7120: In silico and expression analysis.

The high radioresistance of Nostoc sp. strain PCC7120 is indicative of a robust DNA repair pathway. In the absence of NHEJ pathway and the canonical RecBCD proteins, the RecF pathway proteins are expected to play an important role in double strand break repair in this organism. The RecF, RecO and RecR proteins which are central to the RecF pathway have not been characterised in the ancient cyanobacteria, several of which are known to be radioresistant. The characterisation of these proteins was initiated through a mix of in silico, expression and complementation analysis. Differential expression of the recF, recO and recR genes was observed both at the transcript and the protein level under normal growth condition, which did not change significantly upon exposure to DNA damage stresses. Expression of RecR as a 23kDa protein in vivo in Nostoc PCC7120 confirmed the re-annotation of the initiation codon of the gene (alr4977) to a rare initiation codon 'GTT' 267 bases upstream of the annotated initiation codon. Of the three proteins, Nostoc RecO and RecR proteins could complement the corresponding mutations in Escherichia coli, but not RecF. The Nostoc RecO protein exhibited low sequence and structural homology with other bacterial RecO protein, and was predicted to have a longer loop region. Phylogenetic as well as sequence analysis revealed high conservation among bacterial RecR proteins and least for RecO. In silico analysis revealed a comparatively smaller interactome for the Nostoc RecF, RecO and RecR proteins compared to other bacteria, with RecO predicted to interact with both RecF and RecR. The information gathered can form a stepping stone to further characterise these proteins in terms of deciphering their interactome, biochemical and physiological activities. This would help in establishing their importance in RecF pathway of DSB repair in Nostoc PCC7120.

Events associated with DNA replication disruption are not observed in hydrogen peroxide-treated Escherichia coli.

UV irradiation induces pyrimidine dimers that block polymerases and disrupt the replisome. Restoring replication depends on the recF pathway proteins which process and maintain the replication fork DNA to allow the lesion to be repaired before replication resumes. Oxidative DNA lesions, such as those induced by hydrogen peroxide (H2O2), are often thought to require similar processing events, yet far less is known about how cells process oxidative damage during replication. Here we show that replication is not disrupted by H2O2-induced DNA damage in vivo. Following an initial inhibition, replication resumes in the absence of either lesion removal or RecF-processing. Restoring DNA synthesis depends on the presence of manganese in the medium, which we show is required for replication, but not repair to occur. The results demonstrate that replication is enzymatically inactivated, rather than physically disrupted by H2O2-induced DNA damage; indicate that inactivation is likely caused by oxidation of an iron-dependent replication or replication-associated protein that requires manganese to restore activity and synthesis; and address a long standing paradox as to why oxidative glycosylase mutants are defective in repair, yet not hypersensitive to H2O2. The oxygen-sensitive pausing may represent an adaptation that prevents replication from occurring under potentially lethal or mutagenic conditions.

Allosteric effects of SSB C-terminal tail on assembly of E. coli RecOR proteins.

Escherichia coli RecO is a recombination mediator protein that functions in the RecF pathway of homologous recombination, in concert with RecR, and interacts with E. coli single stranded (ss) DNA binding (SSB) protein via the last 9 amino acids of the C-terminal tails (SSB-Ct). Structures of the E. coli RecR and RecOR complexes are unavailable; however, crystal structures from other organisms show differences in RecR oligomeric state and RecO stoichiometry. We report analytical ultracentrifugation studies of E. coli RecR assembly and its interaction with RecO for a range of solution conditions using both sedimentation velocity and equilibrium approaches. We find that RecR exists in a pH-dependent dimer-tetramer equilibrium that explains the different assembly states reported in previous studies. RecO binds with positive cooperativity to a RecR tetramer, forming both RecR4O and RecR4O2 complexes. We find no evidence of a stable RecO complex with RecR dimers. However, binding of RecO to SSB-Ct peptides elicits an allosteric effect, eliminating the positive cooperativity and shifting the equilibrium to favor a RecR4O complex. These studies suggest a mechanism for how SSB binding to RecO influences the distribution of RecOR complexes to facilitate loading of RecA onto SSB coated ssDNA to initiate homologous recombination.

DNA repair pathways important for the survival of Escherichia coli to hydrogen peroxide mediated killing.

Escherichia coli exposed to 1-3mM hydrogen peroxide undergo killing which is designated as the mode-one killing which is a result of oxidative DNA damage. Oxidative stress mediated DNA damage can be repaired by various DNA repair pathways like base excision repair, nucleotide excision repair and homologous recombination repair. In this study we have investigated the role of multiple DNA repair pathways in survival to oxidative killing and assessed their relative importance. Results show that both nucleotide excision repair pathway as well as the RecF pathway of recombination repair are important for repair of the DNA damage caused by exposure to hydrogen peroxide. The study also provides the evidence that RecG helicase which is known for the resolution of Holliday junction intermediates plays a critical role in the survival of mode-one killing by peroxide. There is a severe impact on the survival of repair mutants when parameters like aeration and growth medium are changed. Low aeration and growth in minimal medium provide significant protection from the mode-one killing suggesting that under natural conditions Escherichia coli cells are likely to be protected from the oxidative stress mediated DNA damage.

The DUF328 family member YaaA is a DNA-binding protein with a novel fold

DUF328 family proteins are present in many prokaryotes; however, their molecular activities are unknown. The Escherichia coli DUF328 protein YaaA is a member of the OxyR regulon and is protective against oxidative stress. Because uncharacterized proteins involved in prokaryotic oxidative stress response are rare, we sought to learn more about the DUF328 family. Using comparative genomics, we found a robust association between the DUF328 family and genes involved in DNA recombination and the oxidative stress response. In some proteins, DUF328 domains are fused to other domains involved in DNA binding, recombination, and repair. Cofitness analysis indicates that DUF328 family genes associate with recombination-mediated DNA repair pathways, particularly the RecFOR pathway. Purified recombinant YaaA binds to dsDNA, duplex DNA containing bubbles of unpaired nucleotides, and Holliday junction constructs in vitro with dissociation equilibrium constants of 200-300 nm YaaA binds DNA with positive cooperativity, forming multiple shifted species in electrophoretic mobility shift assays. The 1.65-Å resolution X-ray crystal structure of YaaA reveals that the protein possesses a new fold that we name the cantaloupe fold. YaaA has a positively charged cleft and a helix-hairpin-helix DNA-binding motif found in other DNA repair enzymes. Our results demonstrate that YaaA is a new type of DNA-binding protein associated with the oxidative stress response and that this molecular function is likely conserved in other DUF328 family members.

Structural and functional characterization of oligomeric states of proteins in RecFOR pathway

RecFOR pathway is the principal repair pathway for double strand break and single strand gap repair in Thermus thermophilus. RecF and RecR exist as monomer and dimer in solution, interestingly; they undergo condition-dependent dimerization and tetramerization, respectively during the DNA break repair. However, their importance in protein-protein and protein-DNA interactions remains elusive. In this study, the three-dimensional crystal structures of the wild type RecF and RecR proteins are determined. Thereafter, the structural information is used to mutate the interface residues to cysteine to stabilize the dimeric and tetrameric states of the RecF and RecR proteins, respectively. A comparative study for their interactions with other cognate proteins and ssDNA in native and SSB (single strand binding protein) bound states was performed. RecF or RecFR complex displays a negligible affinity towards ssDNA. Conversely, the RecF mutants and its complexes with wild type RecR showed affinity towards ssDNA, suggesting, distinct modes of interaction of RecF and RecFR complex for ssDNA binding. In the presence of RecO, the stabilized tetrameric RecR showed a lower binding affinity for ssDNA as compared to the SSB bound ssDNA, indicating the importance of tetrameric RecR in stabilizing the RecOR complex on the SSB coated ssDNA. This provides an insight into the reduction of the binding affinity of SSB proteins with the ssDNA, which in turn enhances the recruitment of RecA for strand exchange.

Single-molecule observation of ATP-independent SSB displacement by RecO in Deinococcus radiodurans.

Deinococcus radiodurans (DR) survives in the presence of hundreds of double-stranded DNA (dsDNA) breaks by efficiently repairing such breaks. RecO, a protein that is essential for the extreme radioresistance of DR, is one of the major recombination mediator proteins in the RecA-loading process in the RecFOR pathway. However, how RecO participates in the RecA-loading process is still unclear. In this work, we investigated the function of drRecO using single-molecule techniques. We found that drRecO competes with the ssDNA-binding protein (drSSB) for binding to the freely exposed ssDNA, and efficiently displaces drSSB from ssDNA without consuming ATP. drRecO replaces drSSB and dissociates it completely from ssDNA even though drSSB binds to ssDNA approximately 300 times more strongly than drRecO does. We suggest that drRecO facilitates the loading of RecA onto drSSB-coated ssDNA by utilizing a small drSSB-free space on ssDNA that is generated by the fast diffusion of drSSB on ssDNA.

Assembly and Binding of E. coli RecOR Proteins to SSB C-Terminal Tails

The E. coli single-stranded DNA binding (SSB) protein interacts with at least 17 different proteins, known as SSB-interacting proteins (SIPs), during DNA replication, repair, and recombination. The E. coli RecO protein is a recombination mediator protein functioning in complex with RecF and RecR proteins in the RecF pathway of homologous recombination. RecO has been shown to interact with the last 9 amino acids of the intrinsically disordered C-terminal tails of SSB (SSB-Ct). Although structures of RecOR complexes from organisms, such as D. Radiodurans and T. Tencongensis, have been determined, they differ in stoichiometry. Furthermore, structures of the E. coli RecOR complex are not yet available. We therefore investigated the assembly states of RecO and RecR, and RecOR complexes using analytical ultracentrifugation. We find that E. coli RecO is a stable monomer. Although E. coli RecR had previously been reported to form a dimer, we find that it is in a pH-dependent dimer-tetramer equilibrium. We also find that only the RecR tetramer and not the dimer binds RecO and that the SSB-C-terminal peptide destabilizes the RecOR4 complex (supported by NIH GM030498 to TML).

Features of DNA Helicase Encoded by the uvrD Gene of Deinococcus radiodurans R1 in Escherichia coli K-12 Cells

Reparative helicase II of Deinococcus radiodurans performs an unexpected critical function in repair of double-strand DNA breaks through the mechanism of extended synthesis-dependent strand annealing (ESDSA), while it is considered as an optional participant in the RecF pathway of recombinational repair in Escherichia coli. A fragment of genomic DNA of the radioresistant bacterium Deinococcus radiodurans with the uvrD gene encoding DNA helicase II, which is involved in excision repair of nucleotides, mismatch repair, and recombinational repair and replication, was cloned in the cells of the model object, Escherichia coli K-12. The pCR 2.1-uvrD+ plasmid restores resistance to ultraviolet light of mutant cells of Escherichia coliuvrD–, helD–, and rep– defective in reparative helicase II, helicase IV, and replicative helicase Rep, respectively, almost to the level of wild-type AB1157 and uvrD+, and, to a lesser extent, the strain with a mutation in the recQ gene encoding the key helicase of recombinational repair RecQ. The protective effect is also noticeable when strains with the plasmid are irradiated with γ-rays. It is established that Deinococcus radiodurans UvrD helicase possesses broad possibilities.

Crystal structure of RecR, a member of the RecFOR DNA-repair pathway, from Pseudomonas aeruginosa PAO1.

DNA damage is usually lethal to all organisms. Homologous recombination plays an important role in the DNA damage-repair process in prokaryotic organisms. Two pathways are responsible for homologous recombination in Pseudomonas aeruginosa: the RecBCD pathway and the RecFOR pathway. RecR is an important regulator in the RecFOR homologous recombination pathway in P. aeruginosa. It forms complexes with RecF and RecO that can facilitate the loading of RecA onto ssDNA in the RecFOR pathway. Here, the crystal structure of RecR from P. aeruginosa PAO1 (PaRecR) is reported. PaRecR crystallizes in space group P6122, with two monomers per asymmetric unit. Analytical ultracentrifugation data show that PaRecR forms a stable dimer, but can exist as a tetramer in solution. The crystal structure shows that dimeric PaRecR forms a ring-like tetramer architecture via crystal symmetry. The presence of a ligand in the Walker B motif of one RecR subunit suggests a putative nucleotide-binding site.

Allosteric Effect of E. coli SSB C-Terminal Tails on RecOR Binding to DNA

The E. coli single-stranded DNA binding (SSB) protein interacts with at least 15 different proteins, known as SSB-interacting proteins (SIP), during DNA replication, repair, and recombination. However, the specificity by which SSB differentiates and recruits each SIP for different roles is not understood. The E. coli RecO protein is a recombination mediator protein (RMP) involved in the RecF pathway of homologous recombination. RecO forms complexes with RecF and RecR, and is thought to interact with the last 9 amino acids of the intrinsically disordered C-terminal tails of SSB (SSB-Ct). We are examining the interaction of SSB with RecOR by monitoring the change in intrinsic Trp fluorescence of RecO. We observe an allosteric effect of the SSB-Ct on DNA binding by RecOR, such that the SSB-Ct peptide enhances RecOR binding to ssDNA. Whereas structures of RecOR complexes from other organisms, such as D. Radiodurans and T. Tengcongensis, have been determined, they differ in stoichiometry. Furthermore, structures of the E. coli RecOR complex are not available. We therefore are investigating the functional oligomeric state of RecO and RecR, and the stoichiometry of the RecOR complex using analytical ultracentrifugation and isothermal titration calorimetry. E. coli RecR had previously been reported to form a dimer, but we find that it is in a dimer-tetramer equilibrium and the RecR tetramer appears to be the form that binds RecO (supported by NIH GM030498 to TML).

ATP-dependent conformational change in ABC-ATPase RecF serves as a switch in DNA repair

RecF is a principal member of the RecF pathway. It interacts with RecO and RecR to initiate homologous recombination by loading RecA recombinases on single-stranded DNA and displacing single-stranded DNA-binding proteins. As an ATP-binding cassette ATPase, RecF exhibits ATP-dependent dimerization and structural homology with Rad50 and SMC proteins. However, the mechanism and action pattern of RecF ATP-dependent dimerization remains unclear. Here, We determined three crystal structures of TTERecF, TTERecF-ATP and TTERecF-ATPɤS from Thermoanaerobacter tengcongensis that reveal a novel ATP-driven RecF dimerization. RecF contains a positively charged tunnel on its dimer interface that is essential to ATP binding. Our structural and biochemical data indicate that the Walker A motif serves as a switch and plays a key role in ATP binding and RecF dimerization. Furthermore, Biolayer interferometry assay results showed that the TTERecF interacted with ATP and formed a dimer, displaying a higher affinity for DNA than that of the TTERecF monomer. Overall, our results provide a solid structural basis for understanding the process of RecF binding with ATP and the functional mechanism of ATP-dependent RecF dimerization.

Biochemical characterization of Borrelia burgdorferi's RecA protein.

RecA plays key roles in DNA recombination, replication and repair. Mutation of recA in the Lyme disease spirochete, Borrelia burgdorferi, fails to produce some of the phenotypes expected from study of recA mutation in other organisms. ‘Missing’ recA phenotypes include a lack of growth or viability effects, including in the presence of DNA damage, and a lack of a role in vlsE antigenic variation and infectivity. We present a purification and biochemical characterization of recombinant B. burgdorferi RecA protein. We find that B. burgdorferi 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 E. coli LexA repressor. DNA pairing and strand exchange reactions promoted by B. burgdorferi 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 B. burgdorferi 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 B. burgdorferi’s RecA protein to explain the unusual phenotype of recA mutation and suggest that there may be alternative recombinase functions that could explain the ‘missing’ phenotypes.