Structural studies of the post-cleavage complex in V(D)J recombination

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The biochemical and biological description of the RAG1and RAG2 proteins has suffered for lack of structural information. Studies by this group, in collaboration with the groups of Dr. Wei Yang (LMB), Dr. Alasdair Steven (NIAMS), and Dr. Emilios Dimitriadis (NIBIB) have yielded electron microscope pictures of negatively stained samples, good enough for us to construct a fairly detailed model of the signal-end complex (SEC) of the core RAG1 and RAG2 proteins with DNA. Image reconstruction of the EM pictures revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N-termini of RAG1 and RAG2 were found by antibody staining to be positioned at opposing ends of the complex. Atomic force microscopy revealed that the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N-termini. Correlated molecular weight measurements by scanning transmission electron microscopy, light scattering, and other biophysical methods have resolved controversial issues about the composition of this complex, showing that it contains two monomer units each of RAG1 and RAG2, together with two DNA fragments. The observed molecular weight of 500 kD agrees with the expected value for this composition. The molecular model that incorporates all these data shows strong interactions between the two RAG1 units and between RAG1 and RAG2, but not between the RAG2 monomers. All these studies have relied on continuing improvements in the protein chemistry that have led to samples of much higher purity than previously available. Present efforts focus on the greater detail that can be seen in cryo-electron microscopy and immuno-electron microscopy and also to obtain crystallization of the complex. An additional effort has been devoted to identifying other proteins that bind to the RAG1-RAG2 complex in cells. A preliminary list of candidate proteins has been obtained by mass spectrometry, including some interesting DNA repair factors. Present experiments attempt to validate some of these interactions by co-precipitation assays and by cellular recombination assays with targeted inhibitors.