The major objective of this project is to uncover the enzymatic steps involved in various genetic rearrangement reactions and to study the mechanism of action of the enzymes involved. We are particularly interested in gene rearrangements caused by transposon family of movable genetic elements. The mechanisms of the transposition-replication reaction of bacteriophage Mu is studied under this project as a model system. A critical step in the Mu transposition reaction is a pair of DNA strand transfers which generate an intermediate DNA molecule with a branched structure. Efficient formation of this intermediate requires Mu A, Mu B and E. coli HU proteins along with ATP and Mg++. The Mu A protein binds to the Mu and DNA sequences on the donor DNA and makes a pair of single strand cuts to expose the 3' ends of the Mu sequence. This cleaved donor DNA with associated proteins is an active intermediate which completes DNA strand transfer by using a DNA molecule which is bound by Mu B protein as the target. The Mu B protein possesses an ATPase activity which is stimulated by Mu A protein and DNA, and selectively stimulates the utilization of intermolecular target DNA molecules which do not carry Mu end seguences. The proper relative orientation of the two Mu end seguences on the donor molecule is specifically recognized by making use of the energy of DNA supercoiling and a specific geometry of the Mu end DNA segments within the initial synaptic complex. The intermediate DNA molecules can be converted into cointegrates by DNA replication or into simple inserts by nucleolytic cleavages and gap repair. Both of these resolution pathways are supported by an E. coli cell extract and do not require Mu proteins.
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