Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle.
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.
Hozák
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P
CRC Nuclear Structure and Function Research Group, Sir William Dunn School of Pathology, University of Oxford, UK.
Jackson
D A
DA
Cook
P R
PR
D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000502:physiology* / Q000648:ultrastructure*; D002843:Chromatin / Q000502:physiology; D002875:Chromosomes / Q000502:physiology; D004257:DNA Polymerase II / Q000302:isolation & purification; D004261:DNA Replication / Q000502:physiology*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016253:Microscopy, Immunoelectron; D008957:Models, Genetic; D015530:Nuclear Matrix / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen / Q000302:isolation & purification; D016196:S Phase / Q000502:physiology*; D014544:Uridine Triphosphate / Q000031:analogs & derivatives
D001710:Biotin; D002843:Chromatin; D004257:DNA Polymerase II; D003856:Deoxyuracil Nucleotides; D018809:Proliferating Cell Nuclear Antigen; C087624:biotin-16-dUTP; D014544:Uridine Triphosphate