Biophysical characterization of DNA damage proteins from Rice Plant
DNA damage in living cells is repaired by two main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). The proteins involved in HR pathway have been widely studied in eukaryotes. The MRX/N complex which consists of Mre11, Rad50 and Xrs2 in case of yeast and Nbs1 for vertebrates, are the first responders to double strand breaks. This complex scans the DNA double strand for a break, and binds to the broken DNA ends as soon as a break occurs. The Mre11 protein of the MRN complex is highly conserved in eukaryotes. Rad52 (Radiation sensitive 52) belongs to a different group of important genes, and is found to be highly conserved across different species. Until lately, it was believed that RAD52 is absent in plant systems. However, recent genetic studies have pointed out the presence of RAD52 homologues in plants, though these have not yet been characterized biochemically.
In the current study, the biochemical properties of rice OsMre11 and OsRad52 protein were analysed. These proteins were over-expressed in Escherichia coli over expression host cells and the proteins purified. The identity of purified OsMre11 and OsRad52 protein was confirmed via peptide mass fingerprinting. DNA binding assays showed that the purified proteins were able to bind to different forms of DNA. Purified OsMre11 protein exhibited nuclease properties, which was promoted by presence of manganese as a co- factor. Gel filtration and native PAGE analysis indicated that the OsRad52 protein in its native state probably formed an undecameric structure. Purified OsRad52 mediated the renaturation of complementary single strands into duplex DNA via both agarose gel and FRET based assays. Hence concluding that both OsMre11 and OsRad52 proteins, binds to ssDNA/dsDNA for mediating important functions like nucleolytic cleavage and renaturation during homologous recombination. This study represents the first report on biochemical properties of OsMre11 and OsRad52 protein from important crop like rice, which shall help in dissecting the recombination and repair machinery in plant systems.