STUDIES OF IMMUNOGLOBULIN GENE REARRANGEMENT

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During lymphoid development, functional immunoglobulin and T cell receptor genes are assembled from gene segments by a process called V(D)J recombination, which is essential for generating the diversity of the immune response. In the first stage of this reaction, specific double-strand breaks are made at the target sites by the RAG1/RAG2 protein complex. The later steps necessary for rejoining employ many protein factors that are also used for repair of X-ray damage. We continue to investigate both halves of the pathway.1) We previously described a transpositional recombination carried out by the RAG proteins as a side reaction, and suggested this aberrant activity as a possible source of chromosomal translocations in leukemias and lymphomas. We have now used a biochemical assay to show that the RAG proteins are capable of causing such translocations, although this has not yet been demonstrated in vivo.2) The Xrcc4 protein is known to bind to DNA ligase IV and to stimulate its activity in V(D)J recombination or the repair of radiation damage. The crystal structure of the Xrcc4 protein, which has now been determined in collaboration with W. Yang and M. Junop, resembles the SMC family of proteins involved in many other aspects of DNA metabolism. The binding site for DNA ligase IV lies in the long helical stem of Xrcc4.3) The Mre11/Rad50/Nbs1 complex is known to be involved in many types of genetic recombination We have now shown that the Mre11 nuclease can produce the short homologies often found in V(D)J recombination junctions. Mre11 acts as a dimer to bridge two DNA ends, allows them to sense each other's sequence, and the nuclease pauses to allow ligation when a homology is revealed.4) Phosphorylation of the histone variant H2AX is an early event after DNA damage. We have now shown (in collaboration with W. Bonner and E. Rogakou of NCI) that radiation-induced nuclear foci of phosphorylatd H2AX are precursors of foci involving multiple repair factors. This result links initial signaling at the level of chromatin to later repair of DNA.Melek, M., and Gellert, M. (2000). Cell 101, 625-633.Paull, T. T., and Gellert, M. (2000). Proc. Natl. Acad.Sci. U S A 97, 6409-6414.Paull, T.T. et al. (2000) Curr. Biol. 10, 886-895.