Visualization of replication sites in unfixed human cells.
Sites of DNA replication in nuclei are focally concentrated, suggesting that an underlying structure organizes the activity of many polymerases. As fixation could induce aggregation into foci, we examined the distribution of replication sites in unfixed nuclei. HeLa cells were encapsulated in agarose microbeads, permeabilized in a 'physiological' buffer, their DNA polymerizing activity characterized, and replication sites directly labelled by incubation with fluorochrome-dUTP conjugates. Using conventional and digital fluorescence microscopy, 80-250 foci were seen in these unfixed cells. These foci are unlikely to be formed by the aggregation of separate polymerases as most replication activity found in vivo is retained throughout these procedures. Although commonly used fixation methods collapsed or dispersed their periphery, the central core was very stable. Foci remained when approximately 90% chromatin was removed, suggesting they were attached to an underlying structure.
Hassan
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Sir William Dunn School of Pathology, University of Oxford, UK.
Cook
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PR
D016477:Artifacts; D002463:Cell Membrane Permeability / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004339:Drug Compounding; D005456:Fluorescent Dyes; D006367:HeLa Cells / Q000502:physiology / Q000648:ultrastructure*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D008863:Microspheres; D009685:Nuclear Envelope / Q000187:drug effects / Q000502:physiology; D015530:Nuclear Matrix / Q000502:physiology / Q000648:ultrastructure*; D012685:Sepharose
D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; C027078:deoxyuridine triphosphate; D012685:Sepharose; D004259:DNA-Directed DNA Polymerase