Human RAD52 protein has extreme thermal stability

The human RAD52 protein plays an important role in the earliest stages of c hromosomal double-strand break repair via the homologous recombination path way. Individual subunits of RAD52 associate into seven-membered rings. Thes e rings can form higher order complexes. RAD52 binds to DNA breaks, and rec ent studies suggest that the higher order self-association of the rings pro motes DNA end joining. Monomers of the RAD52(1-192) deletion mutant also as sociate into ring structures but do not form higher order complexes. The th ermal stability of wild-type and mutant RAD52 was studied by differential s canning calorimetry. Three thermal transitions (labeled A, B, and C) were o bserved with melting temperatures of 38.8, 73.1, and 115.2 degreesC. The RA D52(1-192) mutant had only two thermal transitions at 47.6 and 100.9 degree sC (labeled B and C). Transitions were labeled such that transition C corre sponds to complete unfolding of the protein. The effect of temperature and protein concentration on RAD52 self-association was analyzed by dynamic lig ht scattering. From these data a four-state hypothetical model was develope d to explain the thermal denaturation profile of wild-type RAD52. The three thermal transitions in this model were assigned as follows. Transition A w as attributed to the disruption of higher order assemblies of RAD52 rings, transition B to the disruption of rings to individual subunits, and transit ion C to complete unfolding. The ring-shaped quatenary structure of RAD52 a nd the formation of higher ordered complexes of rings appear to contribute to the extreme stability of RAD52. Higher ordered complexes of rings are st able at physiological temperatures in vitro.