V(D)J recombination assembles the variable portions of immunoglobulin and T cell receptor genes and is essential for lymphocyte development. Errors in the recombination process can lead to chromosomal translocations and the development of human malignancies, particularly childhood leukemias. The causes of such translocations are thought to be improper targeting of the recombination machinery and the premature release of broken chromosomal ends before they have been properly rejoined. The proteins encoded by the recombination activating genes, RAG1 and RAG2, play central roles in targeting and DNA end rejoining during V(D)J recombination and are therefore important in the generation of translocations. In the first phase of V(D)J recombination, DNA substrate recognition and the production of double strand breaks take place in highly organized nucleoprotein complexes whose integrity and specificity are in large part determined by RAG1 and RAG2, probably in conjunction with the DNA bending protein HMGB1. Little is known about the structure of these complexes or the conformational changes that occur during DNA binding and cleavage. The hypothesis underlying this proposal is that the sequential, properly regulated formation of these complexes requires an orchestrated series of structural changes in both the substrate DNA and the RAG/HMGB1 proteins. We have demonstrated that RAG1 undergoes a conformational change when it binds DNA and have used fluorescence resonance energy transfer (FRET) assays to characterize DNA organization and bending in single substrate complexes as well as in higher order synaptic complexes. We propose to use a combination of biochemical and fluorescence methods to elucidate protein and DNA structural changes that occur during assembly of RAG protein-DNA complexes and to construct initial three- dimensional models of the DNA in these complexes. The structural information will be connected to function (catalysis of DNA cleavage) through the use of altered DNA substrates, mutated RAG proteins, and fluorescently labeled HMGB1. We are particularly interested in understanding the structural underpinnings of the requirement for asymmetric DNA substrates and the molecular rules governing recently discovered restrictions on the rearrangements of endogenous gene segments. We anticipate that these studies will provide insights into how the DNA and proteins communicate with and influence one another to create a complex within which properly coordinated cleavage events can occur. Public health relevance: These experiments study V(D)J recombination, an essential process that occasionally makes mistakes and generates aberrations in human chromosomes during the development of white blood cells known as lymphocytes. These aberrations contribute to the development of certain blood cancers and it is hoped that this work will provide insights into the causes of these mistakes.
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