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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.

Type

Journal article

Journal

J Cell Sci

Publication Date

08/1994

Volume

107 ( Pt 8)

Pages

2191 - 2202

Keywords

Biotin, Cell Membrane Permeability, Cell Nucleus, Chromatin, Chromosomes, DNA Polymerase II, DNA Replication, Deoxyuracil Nucleotides, HeLa Cells, Humans, Microscopy, Immunoelectron, Models, Genetic, Nuclear Matrix, Proliferating Cell Nuclear Antigen, S Phase