Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblasts

• Published in: Journal of cell science Vol. 99; no. 2; pp. 247 - 253
• Main Authors: FOX, M. H, ARNDT-JOVIN, D. J, JOVIN, T. M, BAUMANN, P. H, ROBERT-NICOUD, M
• Format: Journal Article
• Language: English
• Published: Cambridge Company of Biologists 01.06.1991
• Subjects: Animals; Antibodies, Monoclonal; Biological and medical sciences; Bromodeoxyuridine - metabolism; Cell Line; DNA Replication; Fibroblasts - metabolism; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Image Processing, Computer-Assisted; Mice; Molecular and cellular biology; Molecular genetics; Photomicrography; Replication; S Phase; Cell culture; Confocal system; Antibody; Rodentia; 3T3-Cell line; Site specificity; Vertebrata; Image analysis; Optical microscopy; Mammalia; Three dimensional representation; Mouse; DNA; Replication; Immunofluorescence; Localization; Fibroblast
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.99.2.247

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.