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.

[1]  R Berezney,et al.  Mapping replicational sites in the eucaryotic cell nucleus , 1989, The Journal of cell biology.

[2]  T. Neff,et al.  Effects of antileukemia agents on nuclear matrix-bound DNA replication in CCRF-CEM leukemia cells. , 1988, Cancer research.

[3]  C. Schildkraut,et al.  Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication , 1989, Molecular and cellular biology.

[4]  R. Moyzis,et al.  The human telomere. , 1991, Scientific American.

[5]  H Nakamura,et al.  Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus. , 1986, Experimental cell research.

[6]  S. Avrameas,et al.  Une technique immunoenzymatique pour la mise en évidence de l'hybridation moléculaire entre acides nucléiques , 1983 .

[7]  R. Berezney,et al.  Dynamic association of replicating DNA fragments with the nuclear matrix of regenerating liver. , 1981, Experimental cell research.

[8]  C. Schildkraut,et al.  The temporal order of replication of some DNA cistrons. , 1974, Cold Spring Harbor symposia on quantitative biology.

[9]  D. Jackson,et al.  A cell-cycle-dependent DNA polymerase activity that replicates intact DNA in chromatin. , 1986, Journal of molecular biology.

[10]  J. Bedford,et al.  Comparison of synchronized Chinese hamster ovary cells obtained by mitotic shake-off, hydroxyurea, aphidicolin, or methotrexate. , 1987, Cytometry.

[11]  J. White,et al.  Replication occurs at discrete foci spaced throughout nuclei replicating in vitro. , 1989, Journal of cell science.

[12]  D. Comings,et al.  Initiation of deoxyribonucleic acid replication at the nuclear membrane in human cells. , 1968, Journal of molecular biology.

[13]  T. Taguchi,et al.  Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase-α , 1978, Nature.

[14]  J. Gray,et al.  Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining. , 1988, Cytometry.

[15]  J W Sedat,et al.  Spatial organization of chromosomes in the salivary gland nuclei of Drosophila melanogaster , 1986, The Journal of cell biology.

[16]  J. Huberman,et al.  University of Groningen Organization of Replication of Ribosomal DNA in Saccharomyces cerevisiae , 2017 .

[17]  T M Jovin,et al.  Luminescence digital imaging microscopy. , 1989, Annual review of biophysics and biophysical chemistry.

[18]  J. Tooze,et al.  A monoclonal antibody which recognises each of the nuclear lamin polypeptides in mammalian cells. , 1983, The EMBO journal.

[19]  C. Cornelisse,et al.  Subdivision of S-phase by analysis of nuclear 5-bromodeoxyuridine staining patterns. , 1989, Cytometry.

[20]  L. Deaven,et al.  The temporal structure of S phase , 1975, Cell.

[21]  B. Stillman.  Initiation of eukaryotic DNA replication in vitro , 1988, Annual review of cell biology.

[22]  T M Jovin,et al.  Fluorescence labeling and microscopy of DNA. , 1989, Methods in cell biology.

[23]  T. Ternynck,et al.  Mapping of the pattern of DNA replication in polytene chromosome from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies. , 1985, Cytometry.

[24]  T. Jovin,et al.  Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope , 1990, Journal of microscopy.

[25]  G. Holmquist,et al.  Replication time of interspersed repetitive DNA sequences in hamsters. , 1986, Biochimica et biophysica acta.

[26]  S. Latt.  Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[27]  R A Laskey,et al.  S phase of the cell cycle. , 1989, Science.

[28]  C. H. Ockey,et al.  Distribution of DNA replicator sites in mammalian nuclei after different methods of cell synchronization. , 1970, Experimental cell research.

[29]  H. Gratzner,et al.  Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. , 1982, Science.

[30]  R. Bravo,et al.  Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites , 1987, The Journal of cell biology.

[31]  C. Schildkraut,et al.  Replication program of active and inactive multigene families in mammalian cells , 1988, Molecular and cellular biology.

[32]  R. Hand.  Eucaryotic DNA: Organization of the genome for replication , 1978, Cell.

[33]  R. Umek,et al.  New beginnings in studies of eukaryotic DNA replication origins. , 1989, Biochimica et biophysica acta.

[34]  Thorsten Schormann,et al.  Optical sectioning with a fluorescence confocal SLM: procedures for determination of the 2‐D digital modulation transfer function and for 3‐D reconstruction by tessellation , 1990 .

[35]  J. Huberman,et al.  DNA Replication Sites within Nuclei of Mammalian Cells , 1973, Nature.

[36]  M. Gefter,et al.  DNA Replication , 2019, Advances in Experimental Medicine and Biology.

[37]  M. A. Goldman,et al.  Replication timing of genes and middle repetitive sequences. , 1984, Science.

[38]  G. Holmquist,et al.  Characterization of Giemsa dark- and light-band DNA , 1982, Cell.

[39]  J. Popowski,et al.  Temporal order of gene replication in Chinese hamster ovary cells , 1989, Molecular and cellular biology.

[40]  H. Crissman,et al.  Synchronization of human diploid fibroblasts at multiple stages of the cell cycle. , 1988, Experimental cell research.

[41]  D. Housman,et al.  Changes in the rate of DNA replication fork movement during S phase in mammalian cells. , 1975, Journal of molecular biology.

[42]  N. Nanninga,et al.  Three‐Dimensional Imaging by Confocal Scanning Fluorescence Microscopy a , 1986, Annals of the New York Academy of Sciences.