Genes have to be replicated before every cycle of cell division. Although DNA polymerase has a proofreading mechanism to minimize the errors during replication, occasionally mismatch due to replication-errors still happens. In all living organisms there are mismatch repair systems to prevent such mutations from occurring. E. coli has a methyl-directed mismatch repair system comprising MutS, MutL and MutH proteins. Homologues of MutS and MutL proteins are also found in human. Mutations in these proteins are identified in 90% of the hereditary nonpolyposiscolorectal cancers. In the two years before Oct. 1999, our group determined the crystal structures of mismatch repair proteins MutH and a conserved 40KD fragment of MutL, discovered an intrinsic ATPase activity of MutL, which had eluded scientists for years, and pursued functional studies by mutagenesis and biochemical means.In collaboration with Dr. Peggy Hsieh's group at NIDDK, we have recently determined the crystal structures of the 190 Kd Taq MutS alone, complexed with DNA, and as a ternary complex with DNA and ADPMg2+. These crystal structures have revealed, (1) the architecture of MutS proteins, (2) how MutS binds an unpaired base, (3) how MutS recognizes a broad range of 'mismatches' without sequence specificity, (4) the components of the ATP binding site and hydrolysis mechanism, (5) how the ATPase and the mismatch binding activities are coordinated in MutS, and (6) the 'hot spots' in human MSH proteins where mutations lead to cancers. To continue the studies of DNA recombination, we have continuted our research on V(D)J recombination. V(D)J gene rearrangement in vertebrates is essential for the maturation of immune systems. It allows 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 that becomes causes of various types of 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 turnon the gene rearrangement in vivo. Dr. Martin Gellert's group 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 tested expression of RAG proteins in E. coli. After finally succeeding in making active RAG-1 in E. coli, in collaboration with Dr. Marjorie Oettinger at Mass. General Hospital, we have found the three conserved acidic residues to be responsible for DNA cleavage activity of RAG1 protein and constitute the active site. We have also cloned, expressed and purified various fragments of RAG1 protein for crystallographic purpose. Eventually we are going to determine the three-dimensional structures of RAG proteins and their complexes with the DNA recognition sequences using x-ray crystallographic techniques.
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