Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.
Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA.
D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000302:isolation & purification; D005814:Genes, Viral; D012313:RNA / Q000302:isolation & purification / Q000502:physiology*; D012367:RNA, Viral / Q000302:isolation & purification / Q000502:physiology*; D013539:Simian virus 40 / Q000502:physiology*; D014779:Virus Replication
D004279:DNA, Viral; C034477:RNA primers; D012367:RNA, Viral; D012313:RNA