Journal of Cell Science, Vol 107, Issue 8 2191-2202, Copyright © 1994 by Company of Biologists

JOURNAL ARTICLES

Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle

P Hozak, DA Jackson and PR Cook
CRC Nuclear Structure and Function Research Group, Sir William Dunn School of Pathology, University of Oxford, UK.

Sites of replication in synchronized HeLa cells were visualized by light and electron microscopy; cells were permeabilized and incubated with biotin-16-dUTP, and incorporation sites were immunolabelled. Electron microscopy of thick resinless sections from which approximately 90% chromatin had been removed showed that most DNA synthesis occurs in specific dense structures (replication factories) attached to a diffuse nucleoskeleton. These factories appear at the end of G1-phase and quickly become active; as S-phase progresses, they increase in size and decrease in number like sites of incorporation seen by light microscopy. Electron microscopy of conventional thin sections proved that these factories are a subset of nuclear bodies; they changed in the same characteristic way and contained DNA polymerase alpha and proliferating cell nuclear antigen. As replication factories can be observed and labelled in 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 concentrated beneath the lamina.
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