Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblasts.
The temporal course of replication monitored by 2- or 5-min pulses of bromodeoxyuridine (BrdUrd) incorporation in synchronized 3T3 cells was mapped by high-resolution light microscopy employing a charge-coupled device (CCD) camera and a confocal laser scanning microscope (CLSM). The cells were labeled simultaneously with monoclonal antibodies directed against BrdUrd and nuclear lamin, and stained with the A+T-specific dye 4',6-diamidino-2-phenylindole (DAPI). Stereoscopic reconstructions of cells showing both the lamin and BrdUrd distributions demonstrate that DNA replication occurs at discrete sites in the nucleus, the locations of which progress through a programmed sequence during S phase. Replication begins in a small number of sites in the interior of the nucleus exclusive of the nuclear membrane and proceeds rapidly in early S phase to encompass a relatively large number of small, discrete sites located throughout the nucleus, with the exception of the condensed heterochromatic regions. Replication is primarily confined to the condensed heterochromatic regions in mid-to-late S phase, and to the nuclear periphery at the end of S phase. These distinctive patterns demonstrate a programmed control of replication sites in the spatial domain in differentiated cell nuclei.
Fox
M H
MH
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, FRG.
Arndt-Jovin
D J
DJ
Jovin
T M
TM
Baumann
P H
PH
Robert-Nicoud
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine / Q000378:metabolism; D002460:Cell Line; D004261:DNA Replication; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted; D051379:Mice; D010784:Photomicrography; D016196:S Phase
D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine