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

Abstract

Sites of replication in synchronized HeLa cells were visualized by light and electron microscopy; cells were perme-abilized and incubated with biotin-16-dUTP, and incorporation sites were immunolabelled. Electron microscopy of thick resinless sections from which ∼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 α and proliferating cell nuclear antigen. As replication factories can be observed and labelled in non-permeabi-lized 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.