We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase a. Several antibodies were identified that coprecipitated T antigen and DNA polymerase a, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase a also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.The initiation of viral DNA replication is an important, temporally regulated process during lytic infection of animal cells by DNA tumor viruses (8,58). Although the mechanisms that mediate DNA synthesis in eucaryotic cells are not well understood, in vitro replication assays for tumor virus DNAs have provided a useful tool for studying these processes. Major advances have been made by in vitro analyses of the biochemical steps leading to replication of the linear, double-stranded genome of adenovirus (7,8,51). However, the protein-priming mechanism used during adenovirus DNA synthesis is unlikely to be a general mechanism for cellular replication.By contrast, the small, circular DNA molecules of simian virus 40 (SV40) are of particular interest because several features suggest that the initiation of SV40 replication mimics cellular chromosomal replication. Bidirectional replication initiates from a single origin on the SV40 DNA, and the chromatin associated with the viral genome is similar to that bound to cellular DNA (13). Also, with the exception of the virus-coded large tumor antigen (T antigen), SV40 replication relies entirely on the cellular DNA replication apparatus.The 82,000-dalton T-antigen phosphoprotein has been implicated in a number of biological functions important for both lytic infection of permissive monkey cells and neoplastic transformation of nonpermissive rodent cells. Some of these functions have been mapped to different portions of the T-antigen gene, suggesting that the gene product contains several polypeptide domains that can act with some degree of independence. Three distinct biochemical activities are carried out by T antigen: (i) specific binding to multiple sites at the SV40 origin (48, 54, 56), (ii) ATP hydrolysis (9,20,57), and (iii) association with a 53,000-dalton host protein (29). The DNA binding activity is directly responsible for repression of early viral RNA synthesis, as demonstrated by in vitro transcription reactions supplemented with purified T antigen (23,44).A number of in vivo studies suggest that both the orig...