Although p48 is the most conserved subunit of mammalian DNA polymerase ␣-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein.Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.The small DNA tumor viruses simian virus 40 (SV40) and mouse polyomavirus (PyV) have provided excellent model systems to study the mechanisms and regulation of eukaryotic DNA replication (reviewed in references 7, 9, 55, and 67). Their DNA replication in vivo and in vitro largely depends on the replication apparatus of their hosts, as only one protein, namely, the multifunctional viral large T antigen (Tag), is supplied by the virus (20,26,49).The establishment of cell-free SV40 and PyV DNA replication systems was a major step towards the purification and characterization of essential host replication factors (67). These studies allowed the understanding of the molecular mechanisms involved in the assembly and progression of replication forks (7,28,67). Along with genetic and other biochemical approaches, these DNA replication systems provided insights into the processes of initiation and elongation (67). Interestingly, several parallels between eukaryotic and prokaryotic DNA duplication emerged which show that central processes of DNA metabolism are in part conserved in all kingdoms (33,60,61). The studies of the polyomaviral systems have allowed the development of a general model for eukaryotic DNA replication. In an initial step, Tag recognizes the replication origin (ori) and forms a double hexameric complex, which causes specific distortions of the double-stranded (ds) DNA (4,15,20,37,49). Subsequently, dsDNA is unwound, and multiprotein complexes are assembled by Tag and host replication proteins, such as eukaryotic single-stranded (ss) DNA-binding protein (replication protein A [RPA]), topoisomerase I (topo I), and DNA polymerase ␣-primase (polprim), are recruited (10,11,17,23,38,53,54). After binding and distortion of the dsDNA, the helicase function of Tag melts and unwinds the ds ori DNA (4, 59). For this function, Tag depends on RPA as a cofactor to stabilize the ssDNA resulting from the helicase activity of Tag (7,29,72). Th...