V(D)J gene rearrangement in vertebrates is essential for the maturation of immune systems. 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 are causes for 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 turn on the gene rearrangement in vivo. Dr. Marty 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 tested expression of RAG proteins in E. coli. After making dozens of different fusion constructs, we have finally succeeded in making active RAG-1 in E. coli, which paves the road for both mutational studies as well as crystallographic studies of this extremely important protein. We have also cloned human RAG proteins and made constructs for expression in both E. coli and insect cells. 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. 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. The homologues of MutS and MutL proteins are also found in human. Mutations in these proteins are culprits in 90% of the hereditary nonpolyposis colorectal cancers. During last year, we have grown crystals of MutH, a sequence-specific endonuclease which is activated by MutS upon its recognition of mismatch. We have obtained two crystal forms of MutH, both diffract x-ray to atomic resolution. Recently we have solved and refined the structures of MutH, a single polypeptide chain of 229 aa, in three different crystal packing environments. We have also obtained crystals of MutL and are currently in the process of solving its structure.
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