Polyoma virus minichromosomes: a soluble in vitro replication system.

Polyomavirusminichromosomes were isolated frominfected 3T6cellsby hypotonic extraction ofisolated nuclei. Thekinetics ofinvitro DNA synthesis in thenuclear extract was similar tothatobserved withintact nuclei. Themajority oftheproducts ofinvitro DNA synthesis sedimented withreplicative intermediate (RI)minichromosomes andmigrated astwobands(RI-a andRI-b) on 1.4% agarosegels. Thekinetics ofdeoxynucleotide monophosphate incorporation into these species was consistent withtheexistence ofseveral rate-limiting stepsinin vitro replication bypolyomaminichromosomes. Electron microscope analysis showedthattheRI-abandconsisted almost entirely ofRI structures ranging from46to87%replicated, withone-half ofall structures 67+ 4%replicated. TheRI-bmaterial was more complex, consisting ofa anda structures withtails ranging from7to114%ofpolyoma genome length and,less frequently, oflinked andmultiply linked dimeric structures. Asafoundation fortheeventual reconstitutionofchromatin replication invitro, wehave studied thereplication ofisolated polyoma minichromosomes. Thesesmall, well-defined viral genomeshaveachromatin structure similar to thatofthehost(3, 7,20)andprovide anexcellent modelforeucaryotic replication (5,22,28,29). Withtheexception ofthevirus-coded Tantigen, whichisresponsible forinitiating eachnew roundofDNA replication (26), thehostprovides allofthemachinery necessary forviral replication. Inthis paper, wehavecharacterized endogenousDNA synthesis byisolated minichromosomesandhaveshowntheaccumulation ofreplicative intermediates (RI-a andRI-b). Inthe second paper(13), theelectron microscope was usedtoquantitate andcharacterize DNA species associated withmature(form I)andreplicating (RI)minichromosomes. Inthethird paper(8), we showthatseveral enzymeactivities implicatedinDNA replication cosediment withpolyomaminichromosomes.