Abstract
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.
Highlights
RecA plays key roles in the processes of homologous recombination, DNA damage sensing/ repair and the resetting of stalled or collapsed replication forks [1,2,3]
The activated form of RecA senses excessive ssDNA production to signal the presence of DNA damage or invading DNA through activation of a specialized DNA damage response referred to as the SOS response
We show that B. burgdorferi RecA possesses many of the expected properties of bacterial RecA’s but find that DNA pairing reactions to be unusually dependent upon the presence of ssDNA binding protein (SSB)
Summary
RecA plays key roles in the processes of homologous recombination, DNA damage sensing/ repair and the resetting of stalled or collapsed replication forks [1,2,3]. The activated form of RecA (the ATP-bound presynaptic filament formed on ssDNA) senses excessive ssDNA production to signal the presence of DNA damage or invading DNA through activation of a specialized DNA damage response referred to as the SOS response. DNA strand exchange promoted by RecA is often described as occurring in three stages: presynapsis, synapsis and branch migration. The presynaptic stage involves ATP-promoted cooperative binding of RecA to ssDNA to produce the presynaptic filament [5,6]. The presynaptic filament promotes all the catalytic steps of strand exchange and possesses coprotease activity. E. coli RecA and many other RecA-like proteins are able to extend strand exchange over long stretches of DNA in a process called branch migration [11]
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