A Single DNA Binding Site of DprA Dimer Is Required to Facilitate RecA Filament Nucleation

DprA (also known as Smf) is a conserved RecA mediator originally characterized by its role in natural chromosomal transformation, yet its widespread presence across bacteria hints at broader DNA metabolic functions. Here, we demonstrate that <i>Bacillus subtilis</i> DprA enhances the frequency of <i>Escherichia coli</i> Hfr conjugation in vivo. In vitro, RecA·ATP binds and cooperatively polymerizes in a 50-nucleotide (nt) polydeoxy T (dT)₅₀ ssDNA to form dynamic filaments that SSB inhibits, an effect fully reversed by <i>Bacillus subtilis</i> DprA. <i>Escherichia coli</i> RecA bound to (dT)₂₁ exhibits minimal dATPase activity, but the addition of <i>B. subtilis</i> DprA significantly stimulates RecA dATP hydrolysis. <i>B. subtilis</i> RecA·dATP readily assembles on (dT)₂₀ complexes, and DprA allosterically activates RecA on even shorter (dT)₁₅ substrates. Combining biochemical assays with a fully atomic model of the RecA-DprA-ssDNA complex, we proposed that only one DNA binding site of the DprA dimer engages the ssDNA during RecA loading, owing to steric constraints. This work refines the mechanism of DprA-mediated RecA nucleation and defines the minimal ssDNA footprint required for mediator activity.