THE MOUSE DNA-POLYMERASE ALPHA-PRIMASE SUBUNIT P48 MEDIATES SPECIES-SPECIFIC REPLICATION OF POLYOMAVIRUS DNA IN-VITRO

Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human cell extracts do not. However, the addition of purified mouse DNA polymerase alpha-primase to human cell extracts renders them permissive for Py DNA replication, suggesting that mouse polymerase alpha-primase determines the species specificity of Py DNA replication. We set out to identify the subunit of mouse polymerase al pha-primase that mediates this species specificity. To this end, we cl oned and expressed cDNAs encoding all four subunits of mouse and human polymerase alpha-primase. Purified recombinant mouse polymerase alpha -primase and a hybrid DNA polymerase alpha-primase complex composed of human subunits p180 and p68 and mouse subunits p58 and p48 supported Py DNA replication in human cell extracts depleted of polymerase alpha -primase, suggesting that the primase heterodimer or one of its subuni ts controls host specificity. To determine whether both mouse primase subunits were required, recombinant hybrid polymerase alpha-primases c ontaining only one mouse primase subunit, p48 or p58, together with th ree human subunits, were assayed for Py replication activity. Only the hybrid containing mouse p48 efficiently replicated Py DNA in depleted human cell extracts. Moreover, in a purified initiation assay contain ing Py T antigen, replication protein A (RP-A) and topoisomerase I, on ly the hybrid polymerase alpha-primase containing the mouse p48 subuni t initiated primer synthesis on Py origin DNA. Together, these results indicate that the p48 subunit is primarily responsible for the specie s specificity of Py DNA replication in vitro. Specific physical associ ation of Py T antigen with purified recombinant DNA polymerase alpha-p rimase, mouse DNA primase heterodimer, and mouse p48 suggested that di rect interactions between Py T antigen and primase could play a role i n species-specific initiation of Py replication.