Function of the RAG 1 Ring Finger

DESCRIPTION (provided by applicant): The adaptive immune system of vertebrates depends upon a DNA recombination system, called V(D)J recombination, to generate the repertoire of immunoglobulin and T-cell receptors. Much of the regulation of this recombination process occurs at the initial stage when the DNA is cut. The proteins RAG1 and RAG2 form the site-specific nuclease that cuts the appropriate DNA targets and, it is believed, coordinate the recombination reaction. The RAG1 protein possesses a large N-terminal domain, containing a ring finger motif, which is expendable with respect to the known enzymatic functions as a DNA binding protein and a nuclease. I propose that this domain plays a regulatory role through an activity as an E3 ligase. This is a proposal for the funding of a pilot study to demonstrate that this domain can assist in the ubiquitylation of an artificial substrate in vitro. If this can be achieved, I will test which of the family of E2 proteins best cooperate in the reaction, and pursue the identification of the natural targets in the cell.