We have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) DNA-binding proteins from
Bacillus subtilis
, SsbA and SsbB. During transformation, SsbB localizes at the DNA entry pole where it binds and protects internalized ssDNA. The 2.8-Å resolution structure of SsbB bound to ssDNA reveals a similar overall protein architecture and ssDNA-binding surface to that of
Escherichia coli
SSB. SsbA, which binds ssDNA with higher affinity than SsbB, co-assembles onto SsbB-coated ssDNA and the two proteins inhibit ssDNA binding by the recombinase RecA. During chromosomal transformation, the RecA mediators RecO and DprA provide RecA access to ssDNA. Interestingly, RecO interaction with ssDNA-bound SsbA helps to dislodge both SsbA and SsbB from the DNA more efficiently than if the DNA is coated only with SsbA. Once RecA is nucleated onto the ssDNA, RecA filament elongation displaces SsbA and SsbB and enables RecA-mediated DNA strand exchange. During plasmid transformation, RecO localizes to the entry pole and catalyzes annealing of SsbA- or SsbA/SsbB-coated complementary ssDNAs to form duplex DNA with ssDNA tails. Our results provide a mechanistic framework for rationalizing the coordinated events modulated by SsbA, SsbB and RecO that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.
Bacillus subtilis
RecU protein is involved in homologous recombination, DNA repair, and chromosome segregation. Purified RecU binds preferentially to three- and four-strand junctions when compared to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) (≈10- and ≈40-fold lower efficiency, respectively). RecU cleaves mobile four-way junctions but fails to cleave a linear dsDNA with a putative cognate site, a finding consistent with a similar genetic defect observed for genes classified within the ε epistatic group (namely
ruv
A,
rec
D, and
rec
U). In the presence of Mg
2+
, RecU also anneals a circular ssDNA and a homologous linear dsDNA with a ssDNA tail and a linear ssDNA and a homologous supercoiled dsDNA substrate. These results suggest that RecU, which cleaves recombination intermediates with high specificity, might also help in their assembly.
Background: Different mediators assist RecA to catalyze genetic recombination. Results: DprA facilitates the displacement of both SSBs (SsbB and SsbA), increases RecA nucleation onto SSB-coated ssDNA, and mediates DNA strand annealing. Conclusion: DprA facilitates RecA-mediated strand exchange and anneals complementary strands coated by an SSB protein.Significance: RecA-dependent chromosomal transformation and RecA-independent plasmid transformation require the competence-induced DprA mediator.
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