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Introduction In 1963, Cairns invented DNA fiber autoradiography to spread and visualize [H]thymidine-labeled chromosomal DNA from Escherichia coli cells (Fig. 1 A). Using this technique, bidirectional replication was found to start at a single site (Cairns, 1963; Prescott and Kuempel, 1972), but its position could not be determined. Meanwhile, genetic studies suggested that in E. coli, each autonomous replication unit (replicon) contained a cisacting element, the replicator, and a trans-acting element, the structural gene for the initiator, whose interaction triggered replication (Jacob et al., 1964). Isolation of the chromosomal replicator (oriC) and initiator (DnaA) followed and led to in vitro reconstitution of replication initiation (Bramhill and Kornberg, 1988). In this reaction, DnaA binds and unwinds oriC to load the replicative helicase DnaB onto single DNA strands, seeding the assembly of two divergent replisomes (Bell and Kaguni, 2013; Costa et al., 2013). When Huberman and Riggs (1968) applied DNA autoradiography to eukaryotic cells, they found that replication started