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 self-associate into rings that can then form higher order complexes. RAD52 binds to double-strand DNA ends, and recent studies suggest that the higher order self-association of the rings promotes DNA end-joining. Earlier studies defined the selfassociation domain of RAD52 to a unique region in the N-terminal half of the protein. Here we show that there are in fact two experimentally separable self-association domains in RAD52. The N-terminal self-association domain mediates the assembly of monomers into rings, and the previously unidentified domain in the C-terminal half of the protein mediates higher order self-association of the rings.The repair of double-strand breaks in chromosomal DNA is of critical importance for the maintenance of genomic integrity. In Saccharomyces cerevisiae, genes of the RAD52 epistasis group, RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2, were identified initially by the sensitivity of mutants to ionizing radiation (1, 2). These genes have been implicated in an array of recombination events including mitotic and meiotic recombination as well as doublestrand break repair. RAD52 mutants show the most severe pleiotropic defects suggesting a critical role for the protein in homologous recombination and double-strand break repair (2). The importance of specific protein-protein interactions in the catalysis of homologous recombination is suggested by studies demonstrating specific contacts and functional interactions between Rad52p and a number of proteins involved in recombination including Rad51p (3-8), which catalyzes homologous pairing and strand exchange, and replication factor A (RPA) 1 (8 -10), a heterotrimeric single-stranded DNA binding protein (11).Studies of the equivalent human proteins have identified similar interactions between the RAD52, RAD51, and replication protein A proteins (12-17). Based on a series of proteinprotein interaction assays (15,16,18) and DNA binding studies 2 (16), a domain map of RAD52 was proposed by Park et al. (16) (see Fig. 1). The determinants of self-association were proposed to exist exclusively within a region defined by residues 65-165, a result supported by recent studies of several isoforms of RAD52 (19). Electron microscopy (EM) studies of Rad52p and RAD52 have revealed formation of ring-shaped structures (9 -13 nm in diameter), as well as higher order aggregates (9,12,20). Stasiak et al. (21) performed image analyses of negatively stained electron micrographs and determined that the 10-nm RAD52 rings are composed of seven subunits. Scanning transmission electron microscopy (STEM) analysis indicated a mean mass of 330 Ϯ 59 kDa supporting a heptameric ring-shaped RAD52 structure (21). Recent studies show that RAD52 binds to double-stranded DNA ends as an aggregated complex (20). These end-binding complexes were amorphous in shape and ranged in ...

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P22 bacteriophage has been studied extensively and has served as a model for many important processes such as in vivo protein folding, protein aggregation and protein-protein interactions. The trimeric tailspike protein (TSP) serves as the receptor-binding protein for the P22 bacteriophage to the bacterial host. The homotrimeric P22 tail consists of three chains of 666aa in which the first 108aa form a trimeric dome-like structure which is called the N-terminal domain (NTD) and is responsible for attachment of the tailspike protein to the rest of the phage particle structure in the phage assembly pathway. Knowledge of this interaction requires information on what amino acids are interacting in the interface and how the NTD structure is maintained. The first 23aa form the "stem peptide" which originates at the dome top and terminates at the dome bottom. It contains a hydrophobic valine patch (V8-V9-V10) located within the dome structure. It is hypothesized that the interaction between the hydrophobic valine patch located on stem peptide and the adjacent polypeptide is critical for the interchain interaction which should be important for the stability of the P22 TSP NTD itself. To test this hypothesis, each amino acid in the valine residues is substituted by an acid, a basic, and a hydrophobic amino acid. The results of such substitutions are presented as well as associated studies. The data strongly suggest that the valine patch is of critical importance in the hydrophobic interaction between stem peptide valine patch and an adjacent chain.

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...

The P22 tailspike protein is an intensely studied protein whose structure and sequence has been described. However, a study, describing important protein interactions related to its function at the N-terminal domain, has been lacking. The P22 tailspike protein (TSP) consists of three identical polypeptide chains of 666aa. The first 108 of the 666aa in the P22 TSP form a trimeric N-terminal domain (NTD). Each of the three chains of the trimeric NTD contributes to the formation of a dome-like structure. Our studies suggest that a short stretch of amino acids located within the first fifteen amino acids of the P22 TSP NTD is critical for the stability of the dome structure formed by the first 108aa of the P22 TSP NTD. The first 23aa are located within this dome-like structure and have been dubbed the "stem" of the NTD. Although amino acid residues in the first 15aa (lower stem) are critical, deletion analysis and in vitro assembly studies implicate the rest of the stem in additional stabilizing interactions. Our studies implicate a common protein-protein interaction motif made up of interchain hydrophobic contacts between adjacent chains