Characterizing the Role of Deinococcus Radiodurans RecN Protein in the RecA-mediated DNA Strand Exchange Reaction in Vitro

The bacterial RecA protein is involved in the faithful repair of DNA double strand breaks (DSBs) via homologous recombination.  Many proteins play a role in facilitating the activation of RecA or the molecular events required of RecA in order to mediate the homologous recombination process.  We have discovered that the Deinococcus radiodurans RecN protein promotes the RecA-mediated DNA strand exchange reaction (homologous recombination in vitro) by h olding DNA molecules close together and through a possible interaction with RecA-ssDNA filaments.  This event facilitates the RecA-dependent invasion and homology search within DNA molecules.  Our lab has established RecN to be an ATPase protein with DNA binding capabilities. To further characterize the function of RecN during the DNA strand exchange reaction catalyzed by RecA, we conducted DNA strand exchange experiments in the presence of a RecN mutant protein (RecN R452A).  The RecN R452A protein was added at different stages of the modular RecA-mediated DNA strand exchange reaction.  In contrast to wild-type RecN, RecN R452A inhibits product formation during the DNA strand exchange when present at stages of the reaction before the addition of linear double stranded DNA.  Also, product formation decreases with increasing concentrations of RecN R452A when added after joint molecule formation.  We provide evidence that the mutant protein binds tighter to double stranded DNA than the wild-type protein.  These observations lead us to speculate that high affinity DNA binding results in RecN inhibition of the RecN inhibition of the RecA-dependent invasion and homology search process.