V(D)J gene rearrangement in vertebrates is essential for the maturation of immune system. It allow the generation of antibodies and T-cell receptors to build up the defense system. Such gene rearrangement has to be tightly controlled during cell development. Erroneous rearrangement often leads to gene truncation or chromosome translocation which often causes various lymphomas. V(D)J gene rearrangement is a type of site-specific DNA recombination. Two proteins, RAG-1 and RAG-2 (recombination activation gene products), are necessary and sufficient to turn on the gene rearrangement in vivo. Dr. Martin Gellert's group here at NIH is the first to demonstrate purified RAG-1 and RAG-2 proteins can initiate gene rearrangement in vitro. Active RAG proteins from mouse have been over-expressed in insect cells. My group has scaled up the protein production. We are currently characterizing both RAG-1 and RAG-2 proteins biochemically and plan to determine the three-dimensional structures of each protein and their complexes with the DNA recognition sequences using x-ray crystallographic techniques. Genes have to be replicated before every cycle of cell division. Although DAN polymerase has a proofreading mechanism to minimize the errors during replication, occasionally mismatch due to replication- errors still happens. In all living organisms there is such a mismatch repair system to prevent mutation from occurring. Dr. Hsieh's group at NIDDK has cloned the mismatch-repair protein MutS and MutL from thermus aquaticus (TAQ) and overexpressed the proteins in E.coli. TAQ proteins are homologues of E.coli proteins and recognize mismatch or 1-3 nucleotide-loopout in DNA duplexes. The homologues of these mismatch-repair proteins are also found in human. Mutations in these proteins are found in 90% of the hereditary nonpolyposis colorectal cancer. We are currently crystallizing TAQ MutS protein and its complexes with abnormal DNAs. Once diffracting crystals are obtained, we will determine the atomic structure by x-ray diffraction method and try to understand the mechanism of mismatch DNA repair.
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