Physical and functional interactions of simian virus 40 (SV40) and polyomavirus large-T antigens with DNA polymerase ox-primase were analyzed to elucidate the molecular basis for the species specificity of polymerase a-primase in viral DNA replication. SV40 T antigen associated more efficiently with polymerase oe-primase in crude human extracts than in mouse extracts, while polyomavirus T antigen interacted preferentially with polymerase a-primase in mouse extracts. The apparent species specificity of complex formation was not observed when purified polymerase ft-primases were substituted for the crude extracts. Several functional interactions between T antigen and purified polymerase ax-primase, including stimulation of primer synthesis and primer elongation on M13 DNA in the presence or absence of the single-stranded DNA binding protein RP-A, also proved to be independent of the species from which polymerase aL-primase had been purified. However, the human DNA polymerase a-primase was specifically required for primosome assembly and primer synthesis on SV40 origin DNA in the presence of T antigen and RP-A.Replication of simian virus 40 (SV40) DNA in infected monkey cells has served as a useful model system for mammalian chromosomal replication (for reviews, see references 1, 10, 19, 34, and 64). The viral genome is complexed with histones as a minichromosome and replicates in the S phase of the cell cycle. Initiation of SV40 DNA synthesis takes place within a short, well-characterized origin region in the viral genome; elongation proceeds bidirectionally from the origin and terminates when the two replication forks meet. Except for a single viral protein, the large tumor (T) antigen, SV40 DNA is replicated by cellular proteins.Biochemical studies of SV40 DNA replication have been facilitated by a cell-free replication assay (38,65,78). Extracts of primate cells, but not of other mammalian cells, were found to support replication of SV40 DNA in vitro, reflecting their viral DNA replication activity in cell culture (39). Fractionation of human cell extracts permitted the isolation of a set of seven cellular proteins that were sufficient to reconstitute viral DNA replication in vitro (35,69,74). Only three of these proteins, DNA polymerase ox-primase, the single-stranded DNA-binding protein RP-A, and topoisomerase I, are sufficient, together with T antigen, to initiate viral DNA replication (9,25,35,41,45,69,75,79 unwinding of the template catalyzed by the DNA helicase activity of T antigen (2,3,8,9,15,16,18,20,54,55,57,63,80). RP-A binds to the parental single strands, topoisomerase I releases torsional stress generated by unwinding, and DNA polymerase oa-primase synthesizes the first primers on both strands. Subsequently, DNA polymerase delta and its accessory proteins assemble at the primer-template junction to form the leading-strand replication complex (69,71,72). DNA polymerase cx-primase and possibly a third DNA polymerase, synthesize the lagging strands (9,52,53,69,75).These events occur in an ordered se...