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DNA replication occurs in mammalian cells at so-called replication foci occupying defined nuclear sites at specific times during S phase. It is an unresolved problem how this specific spatiotemporal organization of replication foci is determined. Another unresolved question remains as to what extent DNA is redistributed during S phase. To investigate these problems, we visualized the replicating DNA and the replication machinery simultaneously in living HeLa cells. Time-lapse analyses revealed that DNA was not redistributed to other nuclear sites during S phase. Furthermore, the results showed that DNA is organized into stable aggregates equivalent to replication foci. These aggregates, which we call sub-chromosomal foci, stably maintained their replication timing from S phase to S phase. During S-phase progression, the replication machinery sequentially proceeded through spatially adjacent sets of sub-chromosomal foci. These findings imply that the specific nuclear substructure of chromosomes and the order of their stable subunits determine the spatiotemporal organization of DNA replication.

Stable chromosomal units determine the spatial and temporal organization of DNA replication