Human RAD52 Protein Has Extreme Thermal Stability

Abstract

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 associate into seven-membered rings. These rings can form higher order complexes. RAD52 binds to DNA breaks, and recent studies suggest that the higher order self-association of the rings promotes DNA end joining. Monomers of the RAD52(1-192) deletion mutant also associate into ring structures but do not form higher order complexes. The thermal stability of wild-type and mutant RAD52 was studied by differential scanning calorimetry. Three thermal transitions (labeled A, B, and C) were observed with melting temperatures of 38.8, 73.1, and 115.2°C. The RAD52(1-192) mutant had only two thermal transitions at 47.6 and 100.9°C (labeled B and C). Transitions were labeled such that transition C corresponds to complete unfolding of the protein. The effect of temperature and protein concentration on RAD52 self-association was analyzed by dynamic light scattering. From these data a four-state hypothetical model was developed to explain the thermal denaturation profile of wild-type RAD52. The three thermal transitions in this model were assigned as follows. Transition A was attributed to the disruption of higher order assemblies of RAD52 rings, transition B to the disruption of rings to individual subunits, and transition C to complete unfolding. The ring-shaped quaternary structure of RAD52 and the formation of higher ordered complexes of rings appear to contribute to the extreme stability of RAD52. Higher ordered complexes of rings are stable at physiological temperatures in vitro.RAD52 1 protein plays a critical role in mitotic and meiotic recombination as well as double-strand break repair (1, 2). On the basis of a series of protein-protein interaction assays and DNA binding studies (3-5), a domain map of human RAD52 (RAD52) was proposed by Park et al. (Figure 1). Electron microscopy (EM) studies of Saccharomyces cereVisiae and human RAD52 have revealed formation of ringshaped structures (9-13 nm in diameter), as well as higher order aggregates (6-8). The RAD52 rings appear to be composed of seven subunits (9). EM studies also showed that RAD52 recognizes and binds to double-stranded DNA ends as an aggregated complex that ranges in size from approximately 15 to 60 nm in diameter (8). This binding promoted end-to-end association between DNA molecules and stimulated the ligation of both cohesive and blunt DNA ends (8). Recently, by studying wild type and two deletion mutants of RAD52 (Figure 1), we demonstrated that the selfassociation domain in the N-terminal half of RAD52 is responsible for ring formation and that elements in the C-terminal half of the molecule participate in the formation of higher order complexes of rings (10).Due to the biological interest of human RAD52 and the apparent biochemical importance of RAD52 self-association in DNA repair, we studied its multiple levels of selfassociation and stability using biophy...

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Disrupting the Constitutive, Homodimeric Protein–Protein Interface in CK2β Using a Biophysical Fragment-Based Approach

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Temperature‐induced selective death of the C‐domain within angiotensin‐converting enzyme molecule

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