SIMIAN-VIRUS-40 ORIGIN-DEPENDENT AND T-ANTIGEN-DEPENDENT DNA-REPLICATION WITH DROSOPHILA FACTORS IN-VITRO

DNA replication of double-stranded simian virus 40 (SV40) origin-conta ining plasmids, which has been previously thought to be a species-spec ific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Dr osophila melanogaster. This reaction is dependent upon both large T an tigen, the SV40-encoded replication initiator protein and DNA helicase , and a functional T-antigen binding site at the origin of DNA replica tion. The efficiency of replication with extracts derived from Drosoph ila embryos is approximately 10% of that observed with extracts prepar ed from human 293 cells. This activity is not a unique property of emb ryonic extracts, as cytoplasmic extracts from Drosophila tissue cultur e cells also support T-antigen-mediated replication of SV40 DNA. By us ing highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the prese nce of Drosophila replication protein A (RP-A; also known gs single-st randed DNA-binding protein), but neither human RP-A nor Escherichia co li single-stranded DNA-binding protein could substitute for Drosophila RP-A. Zn reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymer ase alpha-primase. These results collectively indicate that many of th e specific functional interactions among T antigen, polymerase cu-prim ase, and RP-A are conserved from primates to Drosophila species. Moreo ver, the observation that SV40 DNA replication tan be performed with D rosophila factors provides a useful assay for the study of bidirection al DNA replication in Drosophila species in the context of a complete replication reaction.