S-PHASE PROGRESSION IN SYNCHRONIZED HUMAN-CELLS

S-phase human cells, active DNA polymerases are clustered at morphologically discrete sites-replication factories. As S-phase proceeds, characteristic patterns of DNA synthesis correlate with the appearance of replication factories at the corresponding nuclear sites. The coordination of different phases of this replication program was investigated. Aphidicolin was used to synchronize HeLa cells at the beginning of S-phase and S-phase progression followed on removing the drug. Characteristic features of the S-phase program were not affected by the duration of treatment, implying that each phase of synthesis must complete before the next can begin. Prolonged exposure did not result in the progressive activation of all potential origins. Permeabilized cells labeled in vitro with biotin-dUTP usually displayed the typical early S-phase pattern, but often with sites of reduced activity. A minority of cells contained larger, aphidicolin-induced replication sites consistent with the fusion of adjacent factories. These quickly reverted to normal, once cells resumed growth-emphasizing the dynamic nature of nuclear organization. No apparent biochemical defects were observed when short drug treatments were used. Cells synchronized in G1 and incubated in aphidicolin for 2-4 h contained replication complexes distributed with the characteristic early S-phase pattern. Most DNA polymerases were blocked at authentic sites of initiation and resumed synthesis at the in vivo rate, once aphidicolin was removed. Conditions optimal for the isolation of early S-phase origins of replication are described. (C) 1995 Academic Press, Inc.