Studies Of Immunoglobulin Gene Rearrangement

Diabetes, Digestive, Kidney Diseases

Investigators

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Abstract

V(D)J recombination, the rearrangement of gene segments to assemble intact immunoglobulin and T cell receptor coding regions, is initiated by a recombinase made up of the RAG1 and RAG2 gene products. RAG1/2 introduces double-stranded breaks in the DNA adjacent to the coding segments, and in the process converts the ends of the coding DNA into DNA hairpins, with the two strands covalently joined to each other. Similar hairpins are formed by some related transposases, such as the bacterial Tn5 and Tn10 and the family of eukaryotic hAT transposases. Hairpinning in all these cases requires a large distortion of the DNA, possibly due to extrusion of a base from the double helix and its binding to a tryptophan residue. Using the limited sequence homologies between RAG1/ RAG2 and these other transposases as a guide, we are testing mutations in tryptophans and nearby residues for their effects on V(D)J recombination in vivo and RAG1/RAG2-mediated DNA cleavage in vitro. Some promising candidate mutations have been identified and are being further tested. We have also developed methods for preparing much larger amounts of the RAG1 and RAG2 proteins, in preparation for planned crystallographic studies.