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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, ∼ 150 focal sites of synthesis were visible in each nucleus by light microscopy; they also contained DNA polymerase α and proliferating cell nuclear antigen. Electron microscopy of thick resinless sections from which ∼ 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. © 1993.

Original publication

DOI

10.1016/0092-8674(93)90235-I

Type

Journal article

Journal

Cell

Publication Date

23/04/1993

Volume

73

Pages

361 - 373