The Role of RAG Proteins in Receptor Revision Events

Assembly of the immunoglobulin (Ig) genes is initiated during development of B cells by a process known as V(D)J recombination. This process requires two lymphoid specific factors, RAG1 and RAG2, which are expressed during B cell development in the bone marrow. The RAG proteins bind to conserved DNA sequences (RSS) that flank component gene segments, and catalyze the initial DNA cleavage events of the V(D)J recombination reaction. The expression of the RAG proteins in mature, peripheral B cells is a matter of some controversy at the present time. However, it has recently been found that there is a potential role for the RAG proteins in recombination events in peripheral B cells. Specifically, the first clear examples of peripheral secondary recombination in Ig heavy chain genes has been detected in a subset of human tonsillar B cells, which is referred to as receptor revision. Furthermore, the revision events discovered included partial V_H gene replacement where internal cryptic RSS sites are present at the recombination junction in the V_H gene. Identification of the cryptic RSS sites at the junction indicates a potential role for the RAG proteins in these events. To determine if RAG proteins are capable of catalyzing the secondary recombination reactions, we proposed to first determine whether the RAG proteins bind and cleave the cryptic RSS DNA sequences. In these studies we are collaborating with Dr. J. Donald Capra and Dr. Stephen Stray, a postdoctoral research associate in Dr. Capra's laboratory. The progress to date on this project includes, first, the finding that RAG1 demonstrates a similar binding affinity and specificity for a cryptic RSS site versus a canonical RSS. Second, the RAG proteins appear to catalyze cleavage of a cryptic RSS, leading to extraordinary efficiency in the production of double-stranded DNA breaks. These preliminary results indicate that the RAG proteins are capable of catalyzing the observed receptor revision events, and further experiments are in progress to confirm our findings.