What RecFOR are for: Structure-function studies of recombination mediator proteins

Homologous recombination repair (HRR) is a major error-free DNA repair mechanism. During HRR, the damaged DNA strand is restored by using undamaged homologous double-stranded (ds)DNA as a template. A strand-exchange reaction between these strands is catalyzed by a recombinase protein, which requires being loaded on single-stranded (ss)DNA. The loading process is facilitated by recombination mediator proteins (RMPs) via a mechanism which is not well understood.;Recombination mediation includes steps of damage recognition, recombinase recruitment and induction of damage responses. In bacteria, the RecF pathway mediates repair of stalled replication and ssDNA gaps (SSGs). RecO and RecR recruit the RecA recombinase on ssDNA by overcoming an inhibitory effect of SSB, which protects ssDNA from degradation and illegitimate repair. The presence of RecF allows RecOR to initiate RecA recruitment at the 3' end of the SSG by an unknown mechanism.;We found that RecF of D. radiodurans (Dr) exhibits a high degree of structural and biochemical similarity with the head domain of Rad50, a key eukaryotic protein involved in dsDNA break repair. RecF neither alone nor in complex with RecR preferentially binds to junction DNA. The DNA binding property and ATPase activity of RecF depend on the DNA structure and the presence of RecR. Our findings suggest a model of SSG recognition via a dynamic protein-protein interaction between dsDNA bound RecF and RecOR which interacts with the SSB-ssDNA complex.;RecR is a protein scaffold that binds to RecF, RecO and possibly RecA. It forms a tetrameric clamp-like structure with an unknown function. Our results indicate that this structure is not important in interaction with the DrRecF-dsDNA complex. Moreover, binding of RecR to RecF-dsDNA occurs without significant contribution from RecR-DNA interaction. Another set of RecR mutants, K23A/R27A and K23E/R27E, bind to RecO comparably to the wild type protein, but cannot load RecA. They are deficient in DNA and RecF binding and inhibit RecO-DNA interaction. These results suggest a yet-unknown step in the recombination mediation.;Our findings indicate that initiation of homologous recombination may result from a network of protein-protein and protein-DNA interaction rather than single-step recognition of the damaged DNA structure.