Studies Of Immunoglobulin Gene Rearrangement

Diabetes, Digestive, Kidney Diseases

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Abstract

We continue to study V(D)J recombination, the rearrangement of gene segments that is used to assemble intact immunoglobulin and T cell receptor coding regions. This process is initiated by a recombinase made up of the RAG1 and RAG2 proteins, which act together to introduce double-stranded breaks into the DNA adjacent to the coding segments, and in the process converts the ends of the coding DNA into covalently joined DNA hairpins. Because similar hairpins are formed by some related transposases, such as the large group of eukaryotic hAT transposases. we are following up the analogy with these enzymes, specifically as it applies to the large distortion of DNA structure required to make hairpinning possible. We have identified two tryptophan residues that may be involved in binding a base extruded from the DNA, and are working further to test this model. We are also investigating the binding of metal ions to RAG1. A tightly bound calcium ion appears to be necessary for activity, although calcium by itself does not support enzymatic activity, which requires magnesium or manganese. Other enzymes of this class operate by a two magnesium-ion mechanism, but in this case a third binding site may be involved. Work is also continuing on constructing variants of the RAG1 and RAG2 proteins, to be tested for their ability to crystallize in a specific complex with DNA. In collaboration with a group in the Netherlands, we are also testing for genetic transposition driven by RAG1/2 in mammalian cells. Previous work has shown that RAG1/2 has a transposase activity in vitro, but earlier attempts to find transposition in vivo have failed.