Initiation of SV40 DNA replication in vivo: location and structure of 5' ends of DNA synthesized in the ori region

Abstract

Initiation sites for DNA synthesis were located at the resolution of single nucleotides in and about the genetically defined origin of replication (ori) in replicating SV40 DNA purified from virus-infected cells. About 50% of the DNA chains contained an oligoribonucleotide of six to nine residues covalently attached to their 5' ends. Although the RNA-DNA linkage varied, the putative RNA primer began predominantly with rA. The data reveal that initiation of DNA synthesis is promoted at a number of DNA sequences that are asymmetrically arranged with respect to ori: 5' ends of nascent DNA are located at several sites within ori, but only on the strand that also serves as the template for early mRNA, while 5' ends of nascent DNA with the opposite orientation are located only outside ori on its early gene side. This clear transition between discontinuous (initiation sites) and continuous (no initiation sites) DNA synthesis defines the origin of bidirectional replication at nucleotides 5210--5211 and demonstrates that discontinuous synthesis occurs predominantly on the retrograde arms of replication forks. Furthermore, it appears that the first nascent DNA chain is initiated within ori by the same mechanism used to initiate nascent DNA ("Okazaki fragments") throughout the genome.