This project is designed to provide new insight into the mechanisms by which foreign DNA interacts with host chromosomal DNA. Specifically, we shall study the development of bacteriophage Mu. This virus resembles in many key features the animal DNA tumor viruses, DNA insertion sequences and DNA transposition elements. We will focus on: 1) The mechanism of replication of Mu DNA. a) The product of the initial replication events will be characterized with respect to: conservative vs. semi-conservative segregation of Mu DNA replicas, single stranded vs. double stranded nature of the product of replication, origin and direction of replication b) The phage supplied requirements for Mu DNA replication will be analyzed with respect to: requirement for the termini of Mu DNA, characterization of Mu specified proteins c) The involvement of attachment of Mu DNA to the host cell membrane will be examined 2) The mechanisms involved in integration of Mu DNA. a) Mu mediated association of plasmid DNA with the host chromosome will be analyzed with respect to: covalent integration, non-covalent association, catenane formation b) The quantitation of integration events during Mu development will be studied with respect to: integration at replication forks; fate of Mu DNA copies present in survivors of brief periods of induction c) The properties of replicas integrated during lytic development will be examined 3) Models for the formation of HcDNA (circular molecules containing Mu and host DNA) will be tested; involvement of protein-DNA complexes will be examined 4) Processing and packaging of Mu DNA will be studied, and in vitro systems will be developed for analysis of the mechanisms involved in these processes 5) The formation of mating systems using transmissible plasmids containing deleted Mu prophage will be attempted in bacterial species other than E. coli.
