DNA replication occurs at discrete sites in pseudonuclei assembled from purified DNA in vitro

Bacteriophage lambda DNA is assembled into pseudonuclei in Xenopus egg extract and replicated semiconservatively under temporal cell cycle control. Here, replication is shown to be regulated spatially and to occur at discrete sites that represent clustered replication forks. Clustered forks are visualized by incorporation of biotin-19-dUTP into nascent DNA. Pulse-label experiments show that discrete replication foci persist throughout S phase. We conclude that the organization of replication forks into discrete clusters, previously described in eukaryotic nuclei, is not dependent on specific eukaryotic DNA sequences or on a preexisting inherited chromosomal or nuclear structure.