Human cell extracts support the replication of SV40 DNA, whereas mouse cell extracts do not. Species specificity is determined at the level of initiation of DNA replication, and it was previously found that this requires the large subunit, p180, of DNA polymerase ␣-primase to be of human origin. Furthermore, a functional interaction between SV40 large T antigen (TAg) and p180 is essential for viral DNA replication. In this study we determined that the N-terminal regions of human p180, which contain the TAg-binding sites, can be replaced with those of murine origin without losing the ability to support SV40 DNA replication in vitro. The same substitutions do not prevent SV40 TAg from stimulating the activity of DNA polymerase ␣-primase on single-stranded DNA in the presence of replication protein A. Furthermore, biophysical studies show that the interactions of human and murine DNA polymerase ␣-primase with SV40 TAg are of a similar magnitude. These studies strongly suggest that requirement of SV40 DNA replication for human DNA polymerase ␣ depends neither on the TAg-binding site being of human origin nor on the strength of the binary interaction between SV40 TAg and DNA polymerase ␣-primase but rather on sequences in the C-terminal region of human p180.Polyomavirus DNA replication has been studied extensively because of the ease with which viruses of this family, which include SV40 and mouse polyoma virus (PyV), 1 can be grown in cell culture and moreover because of the availability of an in vitro replication system, which allows detailed investigation of the individual factors that play a role in the replication process (1, 2). Polyomavirus DNA replication has been found to be largely dependent upon factors involved in replication of the host DNA but does contribute one essential trans-acting factor known as large T antigen (TAg) to the replication complex. TAg is responsible for recognition of the viral origin of replication, at which it forms a double hexamer (3). In the presence of the single-stranded DNA-binding protein, RPA (replication protein A), and topoisomerase I, TAg proceeds to unwind the origin DNA and recruits the DNA polymerase ␣-primase heterotetramer, which then initiates bidirectional DNA synthesis (4 -11). DNA polymerase ␣-primase initiates DNA replication through the action of its smallest subunit, p48, which synthesizes RNA primers that are subsequently elongated by the large subunit, p180 (12-15). The bulk of DNA synthesis is, however, carried out by a more processive enzyme complex consisting of DNA polymerase ␦ and its processivity factor, PCNA (16 -18). The transition between DNA synthesis by DNA polymerases ␣ and ␦ is mediated by the PCNA loading factor replication factor C (16, 19). Throughout the viral replication cycle, TAg double hexamers are thought to act as the replicative helicase (20). For a more extensive account of the DNA replication process and its participating factors see Refs. 1, 2, and 21-23. SV40 and PyV are very similar with regard to DNA replication; their respectiv...