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HeLa cells in early S phase were encapsulated in agarose microbeads, permeabilized, and incubated with biotin-11-dUTP in a "physiological" buffer. Sites of DNA synthesis were then immunolabeled. As others have found, approximately 150 focal sites of synthesis were visible in each nucleus by light microscopy; they also contained DNA polymerase alpha and proliferating cell nuclear antigen. Electron microscopy of thick resinless sections from which approximately 90% of the chromatin had been removed revealed a similar number of dense, morphologically discrete ovoid bodies strung along a nucleoskeleton. The ovoids remained morphologically and functionally intact despite the removal of most of the chromatin. After 2.5 min of incubation with biotin-11-dUTP, the incorporated analog was associated only with ovoids; after 5 min it began to spread into the adjacent chromatin, which became extensively labeled after 1 hr. This provides visual evidence for polymerization "factories" fixed to a skeleton, with replication occurring as the template moves through them.

Type

Journal article

Journal

Cell

Publication Date

23/04/1993

Volume

73

Pages

361 - 373

Keywords

Biotin, Cell Membrane Permeability, Cell Nucleus, Chromatin, DNA Polymerase II, DNA Replication, Female, Fluorescent Antibody Technique, HeLa Cells, Humans, In Vitro Techniques, Microscopy, Electron, Nuclear Matrix, Nuclear Proteins, Proliferating Cell Nuclear Antigen, Uridine Triphosphate