Mechanisms of RecA filament nucleation on ssDNA by the DprA protein.

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.