AMPLIFICATION OF BACTERIOPHAGE-MU DNA BY ROLLING CIRCLE DNA-REPLICATION IN-VITRO

When mini-Mu DNA was allowed to transpose and replicate in vitro over a prolonged period, the products consisted not only of simple inserts and cointegrates but also high molecular weight DNA many times the unit length of mini-Mu. This high molecular weight product contained predominantly full-length mini-Mu DNA and relatively little non-Mu DNA (the vector harboring the mini-Mu element and the target for transposition in the reaction system). It arose from rolling circle DNA replication of templates created by intramolecular strand transfer, which is catalyzed by Mu transposition proteins. A donor substrate, which is a supercoiled plasmid bearing a mini-Mu element, gave rise to large amounts of the high molecular weight product provided that the vector segment outside the mini-Mu element was 2 kilobase pairs or more. When a donor substrate had a vector segment of only 600 base pairs, the mini-Mu element first had to transpose to a larger circular target before giving rise to the high molecular weight product. These results suggest a mechanism by which Mu DNA can be amplified for lytic development without transposing multiple times. By establishing a circular template, multiple copies of Mu can be processively generated from a single initiation event.