A New and Rapid Method for Visualizing DNA Replication in Spread DNA by Immunofluorescence Detection of Incorporated 5-Iododeoxyuridine

Tomilin, N.V.; Rosanov, Y u; Зенин, В. В.; Bozhkov, V.M.; Vig, Baldev K.

doi:10.1006/bbrc.1993.1039

Cited by 11 publications

(2 citation statements)

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“…Finally, slides were incubated with 20 M TOTO-3 in PBS ϩ (20 min, 20 Њ C; Molecular Probes, Inc., Eugene, OR), rinsed twice in PBS, and then mounted in Vectashield (Vector Labs, Inc., Burlingame, CA). DNA fibers were denatured directly (2.5 M HCl, 1 h; 20 Њ C) and immunostained with mouse anti-BrdU IgG and donkey anti-mouse IgG conjugated with Cy-3, as described above (see also Tomilin et al, 1993).…”

Section: Immunolabelingmentioning

confidence: 99%

Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells

Jackson

Pombo

1998

The Journal of Cell Biology

765

777

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In proliferating cells, DNA synthesis must be performed with extreme precision. We show that groups of replicons, labeled together as replicon clusters, form stable units of chromosome structure. HeLa cells were labeled with 5-bromodeoxyuridine (BrdU) at different times of S phase. At the onset of S phase, clusters of replicons were activated in each of ∼750 replication sites. The majority of these replication “foci” were shown to be individual replicon clusters that remained together, as stable cohorts, throughout the following 15 cell cycles. In individual cells, the same replication foci were labeled with BrdU and 5-iododeoxyuridine at the beginning of different cell cycles. In DNA fibers, 95% of replicons in replicon clusters that were labeled at the beginning of one S phase were also labeled at the beginning of the next. This shows that a subset of origins are activated both reliably and efficiently in different cycles.The majority of replication forks activated at the onset of S phase terminated 45–60 min later. During this interval, secondary replicon clusters became active. However, while the activation of early replicons is synchronized at the onset of S phase, different secondary clusters were activated at different times. Nevertheless, replication foci pulse labeled during any short interval of S phase were stable for many cell cycles. We propose that the coordinated replication of related groups of replicons, that form stable replicon clusters, contributes to the efficient activation and propagation of S phase in mammalian cells.

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Section: Immunolabelingmentioning

confidence: 99%

Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells

Jackson

Pombo

1998

The Journal of Cell Biology

765

777

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“…The treatment of cells with 0.02 and 0.03% MMS (but not with 0.01% MMS) decreases the rate of movement of replication forks (Merrick et al, 2004), which has been shown by the replicative labeling of DNA fibers (Tomilin et al, 1993;Jackson and Pombo, 1998). Using FRAP assay, we examined whether the mobility of focal GFP-Fen1 is effected by MMS treatment.…”

Section: Transient Decrease Of Mobility Of Gfp-fen1 After Treatment Omentioning

confidence: 99%

High Mobility of Flap Endonuclease 1 and DNA Polymerase η Associated with Replication Foci in Mammalian S-Phase Nucleus

Solovjeva

Svetlova

Sasina

et al. 2005

MBoC

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Originally detected in fixed cells, DNA replication foci (RFi) were later visualized in living cells by using green fluorescent protein (GFP)-tagged proliferating cell nuclear antigen (PCNA) and DNA ligase I. It was shown using fluorescence redistribution after photobleaching (FRAP) assay that focal GFP-PCNA slowly exchanged, suggesting the existence of a stable replication holocomplex. Here, we used the FRAP assay to study the dynamics of the GFP-tagged PCNA-binding proteins: Flap endonuclease 1 (Fen1) and DNA polymerase (Pol). We also used the GFP-Cockayne syndrome group A (CSA) protein, which does associate with transcription foci after DNA damage. In normal cells, GFP-Pol and GFP-Fen1 are mobile with residence times at RFi (t m ) ϳ2 and ϳ0.8 s, respectively. GFP-CSA is also mobile but does not concentrate at discrete foci. After methyl methanesulfonate (MMS) damage, the mobile fraction of focal GFP-Fen1 decreased and t m increased, but it then recovered. The mobilities of focal GFP-Pol and GFP-PCNA did not change after MMS. The mobility of GFP-CSA did not change after UV-irradiation. These data indicate that the normal replication complex contains at least two mobile subunits. The decrease of the mobile fraction of focal GFP-Fen1 after DNA damage suggests that Fen1 exchange depends on the rate of movement of replication forks. INTRODUCTIONIn mammalian cells, DNA replication during S phase is located at distinct nuclear sites. Replication foci (RFi) each contain on average 10 spatially clustered active replication forks that can be visualized using antibodies against incorporated halogenated deoxyuridines (Nakamura et al., 1986;Nakayasu and Berezney, 1989;van Dierendonck et al., 1989;O'Keefe et al., 1992;Tomilin et al., 1995;Jackson and Pombo, 1998). In fixed cells, the RFi colocalize with the Tritoninsoluble form of the replication protein proliferating cell nuclear antigen (PCNA) (Bravo and McDonald-Bravo, 1987), the p70 subunit of replication protein A (RPA70), protein kinase Cdk2 and cyclin A (Cardoso et al., 1993), DNA polymerase ␣ (Hozak et al., 1993), DNA ligase I (Cardoso et al., 1997), and DNA polymerase (Pol) (Kannouche et al., 2001). Distinct nuclear sites accumulate green fluorescent protein (GFP)-tagged DNA ligase I (Cardoso et al., 1997), GFP-PCNA (Leonhardt et al., 2000;Somanathan et al., 2001). The foci containing GFP-RPA34 (Sporbert et al., 2002) or GFP-Pol (Kannouche et al., 2001) also can be observed in normally proliferating living S-phase cells, indicating that the RFi seen in fixed cells do not arise during the fixation procedure but reflect real clustering of replication forks and associated molecular machines in the nucleus.It was initially suggested that the RFi represent specialized subnuclear organelles or nucleoskeleton-attached replication factories in which preassembled holocomplexes (replisomes) duplicate incoming DNA (Hozak et al., 1993). In agreement with this model, recent studies of living cells expressing GFP-tagged PCNA by using the fluorescence redistribution after photob...

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Mapping Sites Where Replication Initiates in Mammalian Cells Using DNA Fibers

Takebayashi

Manders

Kimura

et al. 2001

Experimental Cell Research

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A New and Rapid Method for Visualizing DNA Replication in Spread DNA by Immunofluorescence Detection of Incorporated 5-Iododeoxyuridine

Cited by 11 publications

References 0 publications

Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells

Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells

High Mobility of Flap Endonuclease 1 and DNA Polymerase η Associated with Replication Foci in Mammalian S-Phase Nucleus

Mapping Sites Where Replication Initiates in Mammalian Cells Using DNA Fibers

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