DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts

Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2μm replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500–1000bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.