Genes have to be replicated before every cycle of celldivision. Although DNA polymerase has a proofreading mechanism to minimize the errorsduring replication, occasionally mismatch due to replication- errors still happens. Inall living organisms there are mismatch repair systems to prevent such mutations fromoccurring. 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. During thelast year, we have determined crystal structures of MutH, a 229aasequence-specific endonuclease which is activated by MutS upon its recognition ofmismatch. We have obtained two crystal forms of MutH, and have solved and refinedMutH structures of bothcrystal forms. The crystal structure of MutH allow us toidentify the active site of thisenzyme. We then made point mutations to confirm the catalyticresidues. Based on the crystal structures, we postulate a mechanism for how MutH isactivated. We also identified the structual similarity between MutH and restrictionendonucleases, such asPvuII, EcoRV and Sau3AI and proposed that type II restrictionenzymes are evolved from a common ancestor. We have now determined the crystal structure of a 40KdN-terminal fragment of MutL. MutL and its homologues, although indispensable for DNAmismatch repair from bacterial to human, has no known function prior to our structuralcharacterization. Based on the structural homology of MutL to an ATPase-containing fragment ofDNA gyrase and results we obtained from various functional studies, we conclude thatMutL is an ATPase. We have since shown that ATP-binding induces conformational changes inMutL and such changes areessential for MutLs function in DNA repair. We have made variousmutants which lacksATPase activity and are currently further characerizing theseMutL mutants.To continue the studies of DNA recombination, we have focusedour attention ona bacterial transposase, TN10. We have generated ample amountsof pure transposase, IHF(a cofactor for DNA transposition) and various DNA substrates forcrystallographic studiesof the system. V(D)J gene rearrangement in vertebrates is essential for thematuration of immune systems. It allows the generation of antibodies and T-cellreceptors to build up the defense system. Such gene rearrangement has to be tightly controlledduring cell development. Erroneous rearrangement often leads to gene truncation orchromosome translocation that becomes causes of various types of lymphomas. V(D)J generearrangement 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 Gellerts group at NIH is the first todemonstrate purified RAG-1 and RAG-2 proteins can initiate gene rearrangement in vitro. ActiveRAG proteins from mouse have been over- expressed in insect cells. My group has testedexpression 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 bothmutational studies as well as crystallographic studies of this extremely importantprotein. 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-dimensionalstructures of RAG proteins and their complexes with the DNA recognition sequences using x-raycrystallographic techniques. - crystal, molecular structure, recombination, DNA repair
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