REPLICATION FACTORIES AND NUCLEAR-BODIES - THE ULTRASTRUCTURAL CHARACTERIZATION OF REPLICATION SITES DURING THE CELL-CYCLE

Sites of replication in synchronized HeLa cells were visualized by lig ht and electron microscopy; cells were permeabilized and incubated wit h biotin-16-dUTP, and incorporation sites were immunolabelled. Electro n microscopy of thick resinless sections from which similar to 90% chr omatin had been removed showed that most DNA synthesis occurs in speci fic dense structures (replication factories) attached to a diffuse nuc leoskeleton. These factories appear at the end of G(1)-phase and quick ly become active; as S-phase progresses, they increase in size and dec rease in number like sites of incorporation seen by light microscopy. Electron microscopy of conventional thin sections proved that these fa ctories are a subset of nuclear bodies; they changed in the same chara cteristic way and contained DNA polymerase a and proliferating cell nu clear antigen. As replication factories can be observed and labelled i n non-permeabilized cells, they cannot be aggregation artifacts. Some replication occurs outside factories at discrete sites on the diffuse skeleton; it becomes significant by mid S-phase and later becomes conc entrated beneath the lamina.