SPECIES-SPECIFIC REPLICATION OF SIMIAN-VIRUS-40 DNA IN-VITRO REQUIRESTHE P180 SUBUNIT OF HUMAN DNA-POLYMERASE ALPHA-PRIMASE

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DN A polymerase alpha-primase is the major species-specific factor, we se t out to determine the subunit(s) of DNA polymerase alpha-primase requ ired for this species specificity. Recombinant human, mouse, and hybri d human-mouse DNA polymerase alpha-primase complexes were expressed wi th baculovirus vectors and purified. All of the recombinant DNA polyme rase alpha-primases showed enzymatic activity and efficiently synthesi zed the complementary strand on an M13 single-stranded DNA template. T he human DNA polymerase alpha-primase (four subunits [HHHH]) and the h ybrid DNA polymerase alpha-primase HHMM (two human subunits and two mo use subunits), containing human p180 and p68 and mouse primase, initia ted SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which D NA polymerase alpha-primase was deleted, while MMMM IR I and the MMHH complex did not. To determine whether the human p180 or p68 subunit wa s required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HM MM and MHMM) or three human subunits and one mouse subunit (MHHH and H MHH) were tested for SV40 DNA replication activity. The hybrid complex es HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiati on assay with purified proteins and replicated SV40 DNA in depleted mo use extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.