This project will explore the biochemistry of DNA processing at the transfer origin (oriT) of the F plasmid from Escherichia coli K-12. DNA strand cleavage at oriT is a crucial initial step in the conjugal transfer of the F plasmid, which is related to a variety of conjugative drug resistance plasmids. Specific cleavage at oriT can occur in the absence of conjugation with the aid of two to four tra gene products. The genetic, physical, and biochemical basis for site-specific strand cleavage at oriT will be examined. Oligonucleotide directed mutagenesis will be used to identify functional sub-domains of oriT. Mapping of relevant tra protein binding sites (e.g., for traI and traY gene products) will be performed by using extracts and exonuclease III protection, and by using purified gene products and footprinting. DNA/protein complexes ("relaxation complexes") will be sought, and their composition and biochemical properties will be investigated. Finally, attempts will be made to reconstitute the oriT cleavage system in vitro, and to study any higher order quaternary complexes by electron microscopy. These studies may help to define in general terms the structural rules for DNA/protein interactions and for formation of nucleoprotein structures. The presence of the F factor, a circular DNA plasmid, confers upon E. coli the ability to transfer DNA to recipient cells that lack an F factor. This process, bacterial conjugation, is initiated in the donor by introduction of a break at a specific site within one strand of the double-stranded F factor DNA. One of the ends created by the break is then transferred into the recipient cell. Dr. Deonier will investigate the biochemical mechanism that is responsible for introduction of the break and initiation of DNA transfer. Determination of the exact location of the break and identifying the plasmid encoded gene products that produce it will be major objectives. This project has the potential to provide information on the poorly understood early steps in genetic recombination. //
