ENDONUCLEASES OF INTERRUPTED PROKARYOTIC GENES

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DESCRIPTION: Dr. Belfort discovered the multiply interesting group one introns of prokaryotes in phage T4, and has since studied their occurrence, structure, and mechanisms of splicing and mobility. The bacterial introns are self-splicing, although probably assisted by cofactors in the cell, and they encode endonucleases that effect their mobility through a specific double-strand DNA break-mediated gene conversion event. A further novelty is that some evolutionarily related endonucleases are also self-splicing proteins ("Inteins"), which must splice precisely to maintain the coding region of the genes they inhabit. Study of these subjects will be divided in Dr. Belfort's laboratory in the future between two grants, one for the mechanism of RNA splicing and DNA recombination in mobility, and the second one for endonuclease mechanism and evolution. There is considerable important progress in the first area, but the immediate background to the specific aims of this application is studies of the structure and mechanism of action of two different types of endonucleases, one from T4 and one from an archaebacterium. Dr. Belfort has characterized the domain structure of these enzymes and their DNA binding sites, and has exposed in some detail the specific DNA-protein interactions; for example, the enzyme I-TevI of T4 contacts the minor groove of DNA, like only a limited collection of other DNA-binding proteins. Its DNA binding site has some unusual conformation, and it consists of separate recognition and cleavage regions that may match the domain structure of the proteins. Dr. Belfort's laboratory has produced crystals of the I-DmoI (archaebacterial) enzyme, promising that atomic structures may soon exist, perhaps of enzyme-DNA complex. They have studied evolution of these elements, e.g. finding that the relatedness of the endonucleases and their resident introns and organisms differ, supporting the idea that the endonucleases and introns arose independently. They noted similarity of the targets of endonucleases to the own surrounds, suggesting that endonucleases mediate their own mobility as well as that of the whole intron. There are two main goals of the new grant. 1) To further understand the protein-DNA interactions of endonucleases: stoichiometry, binding details, role of DNA binding in activity, and domains of the enzymes, particularly the function of the conserved amino acid motifs. They will perform biochemical characterization of the enzymes, and provide enzyme for atomic structure determination. 2) To determine if a conserved adjacent endonuclease recognition sequence in the sunY intron has a regulatory function, and to attempt to evolve an intein from a closely related endonuclease.