S-PHASE PROGRESSION IN SYNCHRONIZED HUMAN-CELLS

S-phase human cells, active DNA polymerases are clustered at morpholog ically discrete sites-replication factories. As S-phase proceeds, char acteristic patterns of DNA synthesis correlate with the appearance of replication factories at the corresponding nuclear sites. The coordina tion 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. Character istic features of the S-phase program were not affected by the duratio n of treatment, implying that each phase of synthesis must complete be fore the next can begin. Prolonged exposure did not result in the prog ressive activation of all potential origins. Permeabilized cells label ed in vitro with biotin-dUTP usually displayed the typical early S-pha se pattern, but often with sites of reduced activity. A minority of ce lls contained larger, aphidicolin-induced replication sites consistent with the fusion of adjacent factories. These quickly reverted to norm al, once cells resumed growth-emphasizing the dynamic nature of nuclea r organization. No apparent biochemical defects were observed when sho rt drug treatments were used. Cells synchronized in G1 and incubated i n aphidicolin for 2-4 h contained replication complexes distributed wi th 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 th e isolation of early S-phase origins of replication are described. (C) 1995 Academic Press, Inc.