Visualization of replication factories attached to nucleoskeleton.
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.
Hozák
P
P
Sir William Dunn School of Pathology, University of Oxford, England.
Hassan
A B
AB
Jackson
D A
DA
Cook
P R
PR
D001710:Biotin; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000648:ultrastructure*; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure; D004257:DNA Polymerase II / Q000378:metabolism; D004261:DNA Replication; D005260:Female; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D015530:Nuclear Matrix / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen; D014544:Uridine Triphosphate / Q000378:metabolism
D002843:Chromatin; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D001710:Biotin; D004257:DNA Polymerase II; D014544:Uridine Triphosphate