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Nucleic Acids Research, 1987, Vol. 15, No. 19 7877-7888
© 1987

Articles

Initiation of simian virus 40 DNA replication in vitro:identification of RNA-Primed nascent DNA chains

Janos Taljanidisz1,2, Scott R. Decker2,3, Zong-Sheng Guo2,4, Melvin L. DePamphilis2,4 and Nilima Sarkar1,2,*

1Department of Metabolic Regulation, Boston Biomedical REsearch Institute, Stanford, CA 94305, USA 20 Staniford StreetBoston, MA 02114 2Department of Biological Chemistry, Harvard Meical School, Stanford, CA 94305, USA 20 Staniford StreetBoston, MA 02114 Boston, MA 02115, USA

*To whom correspondence should be addressed

Received May 26, 1987. Accepted September 3, 1987.

Cell-free extracts of simian virus 40(SV40)-infected CV-1 cells can initiate large rumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 orgin of replication(ori). To determine whether or not DNA replication under these conditions involves RANA-priked DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytideine 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 ehich DNA primkase initiates DNa synthesis on that strand of orithat encodes early mRNA.

3Present addresses: Gene-Trak Systems, 31 New York Ave., Framingham, MA 01701,USA

4Department of Cell and Developmental Biology, Roche Institute of MOlecular Biology, Nutley, NJ 07110,USA


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