The major object 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. The reactions studied include: the site specific recombination of bacteriophage lambda, resolution of the Holliday intermediate of general genetic recombination, the transposition-replication reaction of bacteriophage Mu, and the rearrangement reaction of immunoglobulin genes. The most recent developments include the establishment of an in vitro reaction system for the study of replicative transposition of bacteriophage Mu. This in vitro reaction yields both cointegrate products as well as simple insertion products and requires both the A and B gene products of phage Mu along with other bacterial proteins. By making use of this cell free reaction system, under modified reaction conditions we could isolate the transposition reaction into two separate steps. The first step involves a pair of DNA strand transfer reactions which generate an intermediate DNA molecule. The structure of this intermediate has been determined. The formation of the intermediate can be carried out by three purified protein factors; Mu A, Mu B and E. coli HU proteins. The reaction requires a transposon donor molecule with two Mu end sequences in their proper relative orientation to be negatively supercoiled, while the transposition target DNA can be in relaxed form. The intermediate DNA molecules can be converted into cointegrates by DNA replication or into simple inserts by nucleolytic cleavages and repair, in a second reaction, by an E. coli extract without Mu proteins.
