Department of Molecular Genetics and Microbiology
Catalysis; DNA Cleavage; DNA-Binding Proteins; Dimerization; Homeodomain Proteins; Protein Binding; Protein Structure, Quaternary; Substrate Specificity; *Temperature; VDJ Recombinases
Biochemistry, Biophysics, and Structural Biology | Life Sciences | Medicine and Health Sciences
BACKGROUND: Functional immunoglobulin and T cell receptor genes are produced in developing lymphocytes by V(D)J recombination. The initial site-specific DNA cleavage steps in this process are catalyzed by the V(D)J recombinase, consisting of RAG1 and RAG2, which is directed to appropriate DNA cleavage sites by recognition of the conserved recombination signal sequence (RSS). RAG1 contains both the active site and the RSS binding domains, although RAG2 is also required for DNA cleavage activity. An understanding of the physicochemical properties of the RAG proteins, their association, and their interaction with the RSS is not yet well developed.
RESULTS: Here, we further our investigations into the self-association properties of RAG1 by demonstrating that despite the presence of multiple RAG1 oligomers, only the dimeric form maintains the ability to interact with RAG2 and the RSS. However, facile aggregation of the dimeric form at physiological temperature may render this protein inactive in the absence of RAG2. Upon addition of RAG2 at 37 degrees C, the preferentially stabilized V(D)J recombinase:RSS complex contains a single dimer of RAG1.
CONCLUSION: Together these results confirm that the functional form of RAG1 in V(D)J recombination is in the dimeric state, and that its stability under physiological conditions likely requires complex formation with RAG2. Additionally, in future structural and functional studies of RAG1, it will be important to take into account the temperature-dependent self-association properties of RAG1 described in this study.
De, Pallabi; Zhao, Shuying; Gwyn, Lori M.; Godderz, Leann J.; Peak, Mandy M.; and Rodgers, Karla K., "Thermal Dependency of RAG1 Self-association Properties" (2008). Open Access Articles. 2246.