Vaccinia Virus DNA Polymerase Promotes DNA Pairing and Strand-Transfer Reactions

AbstractVaccinia virus infection results in the synthesis of a protein that promotes joint molecule formation and strand-transfer reactionsin 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 transferin vitroin reactions containing single-stranded circular and linear duplex DNAs. The reaction required homologous substrates and magnesium ions and was stimulated by DNA aggregating agents such as spermidine HCl andEscherichia colisingle-strand DNA binding protein. There was no requirement for a nucleoside triphosphate cofactor. The reaction ceased when ∼20% of the double-stranded substrate had been incorporated into joint molecules and required stoichiometric quantities of DNA polymerase (0.5–1 molecules of polymerase per double-stranded DNA end). Electron microscopy showed that the joint molecules formed during these reactions contained displaced strands and thus represented the products of a strand-exchange reaction. We also reexamined the link between replication and recombination using a luciferase-based transfection assay and cells infected with DNA polymeraseCts42mutant viruses. These data substantiate 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