In vitro concatemer formation catalyzed by vaccinia virus DNA polymerase

During poxvirus infection, both viral genomes and transfected DNAs are conv erted into high-molecular-weight concatemers by the replicative machinery. However, aside from the fact that concatemer formation coincides with viral replication, the mechanism and protein(s) catalyzing the reaction are unkn own. Here we show that vaccinia Virus DNA polymerase can catalyze single-st randed annealing reactions in vitro, converting linear duplex substrates in to linear or circular concatemers, in a manner directed by sequences locate d at the DNA ends. The reaction required greater than or equal to 12 bp of shared sequence and was stimulated by vaccinia single-stranded DNA-binding protein (gpl3L). Varying the structures at the cleaved ends of the molecule s had no effect on efficiency. These duplex-joining reactions are dependent on nucleolytic processing of the molecules by the 3'-to-5' proofreading ex onuclease, as judged by the fact that only a 5'-P-32-end label is retained in the joint molecules and the reaction is inhibited by dNTPs. The resultin g concatemers are joined only through noncovalent bonds, but can be process ed into stable molecules in E. coil, if the homologies permit formation of circular molecules. This reaction provides a starting point for investigati ng the mechanism of viral concatemer formation and can be used to clone PCR -amplified DNA. (C) 2000 Academic Press.