Vaccinia virus DNA polymerase promotes DNA pairing and strand-transfer reactions

Vaccinia virus infection results in the synthesis of a protein that promote s joint molecule formation and strand-transfer reactions in vitro. We show here that this activity is also expressed by vaccinia DNA polymerase (gpE9L ). Recombinant vaccinia polymerase was produced using a hybrid Vaccinia/T7 expression system and purified to homogeneity. This protein catalyzed joint molecule formation and strand transfer in vitro in reactions containing si ngle-stranded circular and linear duplex DNAs. The reaction required homolo gous substrates and magnesium ions and was stimulated by DNA aggregating ag ents such as spermidine HCl and Escherichia coil single-strand DNA binding protein. There was no requirement for a nucleoside triphosphate cofactor. T he reaction ceased when similar to 20% of the double-stranded substrate had been incorporated into joint molecules and required stoichiometric quantit ies of DNA polymerase (0.5-1 molecules of polymerase per double-stranded DN A end). Electron microscopy showed that the joint molecules formed during t hese reactions contained displaced strands and thus represented the product s of a strand-exchange reaction. We also reexamined the link between replic ation and recombination using a luciferase-based transfection assay and cel ls infected with DNA polymerase Cts42 mutant viruses. These data substantia te the claim that there exists an inextricable link between replication and recombination in poxvirus-infected cells. Together, these biochemical and genetic data suggest a way of linking poxviral DNA replication with genetic recombination. (C) 1999 Academic Press.