Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy.

The temporal and spatial progression of DNA replication in interphase nuclei of eukaryotic cells has been investigated. Application of a recently developed technique for the immunofluorescence double staining of cell nuclei labelled first with iododeoxyuridine (IdUrd) and subsequently with chlorodeoxyuridine (CldUrd) allows the visualization of two replication patterns in the same nucleus originating from two different periods of the S-phase. We have analysed changes in the three-dimensional replication patterns during the S-phase. To record dual colour three-dimensional images of doubly stained nuclei, a confocal microscope is used. This CSLM is equipped with a specific laser/filter combination to collect both fluorescence signals (FITC and Texas Red) in a single scan, thus precluding pixel shift between the images. A method for the quantitative evaluation of the degree of overlap between DNA regions replicated in two different periods of the S-phase is applied. The results confirm the generally accepted theory that DNA is replicated coordinately in a specific temporal order during the S-phase. The replication time of a DNA domain (i.e. the time between initiation and termination of DNA replication within a domain) at the very beginning of the S-phase was known to be one hour (Nakamura et al., 1986). Our observations show that in the rest of the S-phase, the replication time of a DNA region is also about one hour. We conclude that replicon clusters located in the same region are replicated in the same relatively short period of time. After this period there is no unreplicated DNA left in this region.

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

[2]  D. Jackson.  The organization of replication centres in higher eukaryotes. , 1990, BioEssays : news and reviews in molecular, cellular and developmental biology.

[3]  M. Vitale,et al.  High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains. , 1990, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[4]  R. Berezney,et al.  Nuclear protein matrix: association with newly synthesized DNA. , 1975, Science.

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

[6]  P. Cook.  The nucleoskeleton and the topology of replication , 1991, Cell.

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

[8]  A. Riggs,et al.  On the mechanism of DNA replication in mammalian chromosomes. , 1968, Journal of molecular biology.

[9]  J. Gray,et al.  Double labeling with iodo- and bromodeoxyuridine for cell kinetics studies. , 1989, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

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

[11]  N. Nanninga,et al.  Three‐dimensional imaging in fluorescence by confocal scanning microscopy , 1989, Journal of microscopy.

[12]  S. Fakan,et al.  Localization of newly-synthesized DNA in a mammalian cell as visualized by high resolution autoradiography. , 1974, Experimental cell research.

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

[14]  D. Prescott,et al.  Late S phase cells (Chinese hamster ovary) induce early S phase DNA labeling patterns in G1 phase nuclei. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

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

[16]  J. Aten,et al.  An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA. , 1991, Cytometry.

[17]  A. van Rotterdam,et al.  Spatial topography of a pericentromeric region (1q12) in hemopoietic cells studied by in situ hybridization and confocal microscopy. , 1990, Cytometry.

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

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

[20]  R. Hand.  8 – Eukaryotic Chromosome Replication and Its Regulation , 1979 .

[21]  B. Vogelstein,et al.  A fixed site of DNA replication in eucaryotic cells , 1980, Cell.

[22]  T M Jovin,et al.  Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblasts. , 1991, Journal of cell science.

[23]  R Berezney,et al.  Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix , 1984, The Journal of cell biology.

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

[25]  G. J. Brakenhoff,et al.  Three-dimensional chromatin distribution in neuroblastoma nuclei shown by confocal scanning laser microscopy , 1985, Nature.

[26]  H. Edenberg,et al.  Eukaryotic chromosome replication. , 1975, Annual review of genetics.

[27]  L. Villarreal.  Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control. , 1991, Microbiological reviews.

[28]  P Dean,et al.  Proposed standard for image cytometry data files. , 1990, Cytometry.