Species-specific replication of simian virus 40 DNA in vitro requires the 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 DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse 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.
Institute for Biochemistry, Ludwig-Maximilians-Universität München, Germany.
F
F
Stadlbauer
C
C
Voitenleitner
A
A
Brückner
E
E
Fanning
H P
HP
Nasheuer
D000595:Amino Acid Sequence; D000818:Animals; D019915:DNA Primase; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology*; D013045:Species Specificity; D014779:Virus Replication / Q000502:physiology*
D004279:DNA, Viral; D011994:Recombinant Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases