Biochemical function of RecN protein in bacterial DNA double strand break repair

The efficient repair of DNA double strand breaks (DSBs) is fundamental for genomic intergrity. Vast genetic evidence suggests that the product of the recN gene is involved in the efficient repair of DSBs in bacteria. However, the specific function of the RecN protein in DSB repair is poorly understood. This is in great part due to the limited biochemical information available to help explain the role of RecN in this process. In this work, we use the pure RecN protein from Deinoccocus radiodurans to describe the biochemical properities of RecN so that we can further our understanding of the molecular role of this protein in DSB repair.;Primary sequence analysis of bacterial RecN proteins suggest that RecN is capable of binding and hydrolyzing ATP, as it contains Walker A and Walker B motifs. We show that Dr RecN is able to hydrolyze ATP with a turnover rate of 24 min-1 in the absence DNA. This DNA-independent turnover rate is stimulated by duplex DNA, but not by single-stranded DNA. The stimulation of the DNA-independent turnover rate of Dr RecN by duplex DNA exhibits a dependence on the concentration of Dr RecN. This indicates that RecN-RecN interactions are required for effective ATP hydrolysis and that duplex DNA stabilizes these interactions. Our results confirm that bacterial RecN proteins are ATPases.;In addition, RecN protein shares sequence homology with proteins from Structural Maintenance of Chromosome (SMC) family. SMC proteins are classified as intra- or intermolecular DNA linkers based on their biochemical activities. Here, we show that Dr RecN facilitates intermolecular DNA associations more likely by tethering DNA molecules. This type of activity is similar to that exhibited by an SMC protein classified as an intermolecular DNA linker. We also show that the DNA tethering activity is greatly affected by ATP. ATP seems to increase the affinity of Dr RecN for duplex DNA, as less Dr RecN protein is required to efficiently stimiulated intermolecular DNA interactions in presence of ATP than in absence of the nucleotide.;The fact that Dr RecN is able to facilitate intermolecular DNA associations incited us to evaluate the contribution of Dr RecN to the RecA-mediated DNA strand exchange reaction. Here, we demonstrate that Dr RecN facilitates the strand exchange reaction when catalyzed by Dr RecA, but not when it is catalyzed by E. coli RecA. This observation suggests that Dr RecN and Dr RecA interact specifically. Further analysis of the effect of Dr RecA on the ATPase activity of Dr RecN reveals that a specific interaction between these proteins is likely to exist since Dr RecA, but not E. coli RecA stimulates the ATPase activity of Dr RecN in presence of a duplex DNA substrate.;The biochemical characterization of the Dr RecN protein presented here furthers our understanding of the role of RecN in DSBs repair by homologous recombination. The biochemical characterization of Dr RecN also provides insight to the role of SMC and SMC-type proteins in recombinational repair.