Mechanisms of RecA filament nucleation on ssDNA by the DprA protein.
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The DprA protein has been implicated in the protection of incoming DNA. However, the presence of dprA gene family members, also known as smf, across diverse bacterial species suggests a broader functionality for their gene products. We examined the role of the Escherichia coli DprA/Smf homologue in conjugation. Remarkably, Bacillus subtilis dprA/smf can complement an E. coli dprA mutant, indicating interchangeability of dprA/smf genes between competent and non-competent species in conjugational processes. The DprA protein forms a complex with DNA, facilitating the nucleation of RecA protein filaments onto circular single-stranded DNA coated with SSB protein. To focus on RecA nucleation, we employed short DNA oligonucleotides that restrict RecA-DNA binding but allow for DprA-RecA-DNA binding. Analysis of dATPase activity revealed that RecA-DNA complexes were readily formed only with olig50, while DprA-RecA-DNA complexes were also feasible with olig21. Combining experimental data with a full-atomic model of the RecA-DprA-ssDNA complex’s spatial structure, we proposed a molecular mechanism for DprA-mediated loading of RecA proteins onto ssDNA. Our findings suggest that only one DprA-ssDNA interaction can occur sterically, occupying one strand of ssDNA in the complex.