Nuclear organization of DNA replication in primary mammalian cells

Using methods that conserve nuclear architecture, we have reanalyzed the sp atial organization of the initiation of mammalian DNA synthesis. Contrary t o the commonly held view that replication begins at hundreds of dispersed n uclear sites, primary fibroblasts initiate synthesis in a limited number of foci that contain replication proteins, surround the nucleolus, and overla p with previously identified internal lamin A/C structures. These foci are established in early G(1)-phase and also contain members of the retinoblast oma protein family. Later, in S-phase, DNA replication sites distribute to regions located throughout the nucleus. As this progression occurs, associa tion with the lamin structure and pRB family members is lost. A similar tem poral progression is found in all the primary cells we have examined but no t in most established cell lines, indicating that the immortalization proce ss modifies spatial control of DNA replication. These findings indicate tha t in normal mammalian cells, the onset of DNA synthesis is coordinately reg ulated at a small number of previously unrecognized perinucleolar sites tha t are selected in early G(1)-phase.