Nuclear Matrix-bound Replicational Sites Detected in Situ by 5-bromodeoxyuridine

Overview

Journal Histochemistry

Specialty Biochemistry

Date 1992 Aug 1

PMID 1429014

Citations 10

Authors

L M Neri

G Mazzotti

S Capitani

N M Maraldi

C Cinti

N Baldini

R Rana

A M Martelli

Affiliations

Soon will be listed here.

Abstract

The nuclear matrix was prepared in situ from Swiss 3T3 cells, which were synchronized by contact inhibition and serum starvation and pulse-labelled for very short periods of time with 5-bromodeoxyuridine (5-BrdU). For the first time 5-BrdU has been employed to demonstrate the association of newly synthesized DNA with a nucleoskeleton. Immunofluorescence analysis using a monoclonal antibody to 5-BrdU revealed five different intranuclear staining patterns at different stages of the S phase. These patterns were observed also in intact cells and did not change during the matrix preparation steps which involve extraction with 2 M NaCl and DNase I digestion. Such an observation was also confirmed by spatial confocal microscopy studies. The intensity of fluorescence, which was evaluated by cytofluorometry, increased to reach a maximum during mid-S phase and then decreased. Because no significant difference was found in the time to label residual DNA of different 5-BrdU staining patterns, this strongly suggests that a different number of replicons is activated at different stages of the S phase. These results strengthen the hypothesis that eukaryotic DNA replication occurs in close association with an insoluble protein nuclear skeleton, which determines the three-dimensional spatial organization of chromosome duplication.

Citing Articles

Supervised and unsupervised deep learning-based approaches for studying DNA replication spatiotemporal dynamics.

Ng-Kee-Kwong J, Philps B, Smith F, Sobieska A, Chen N, Alabert C Commun Biol. 2025; 8(1):311.

PMID: 40011665 PMC: 11865476. DOI: 10.1038/s42003-025-07744-2.

DNA replication and the GINS complex: localization on extended chromatin fibers.

Cohen S, Chastain 2nd P, Cordeiro-Stone M, Kaufman D Epigenetics Chromatin. 2009; 2(1):6.

PMID: 19442263 PMC: 2686697. DOI: 10.1186/1756-8935-2-6.

Replication of centromeric heterochromatin in mouse fibroblasts takes place in early, middle, and late S phase.

Weidtkamp-Peters S, Rahn H, Cardoso M, Hemmerich P Histochem Cell Biol. 2005; 125(1-2):91-102.

PMID: 16231189 DOI: 10.1007/s00418-005-0063-3.

Nuclear organization of DNA replication in primary mammalian cells.

Kennedy B, Barbie D, Classon M, Dyson N, Harlow E Genes Dev. 2000; 14(22):2855-68.

PMID: 11090133 PMC: 317063. DOI: 10.1101/gad.842600.

Association of chromosome territories with the nuclear matrix. Disruption of human chromosome territories correlates with the release of a subset of nuclear matrix proteins.

Ma H, Siegel A, Berezney R J Cell Biol. 1999; 146(3):531-42.

PMID: 10444063 PMC: 2150557. DOI: 10.1083/jcb.146.3.531.

References

Van Dierendonck J, Keyzer R, van de Velde C, Cornelisse C . Subdivision of S-phase by analysis of nuclear 5-bromodeoxyuridine staining patterns. Cytometry. 1989; 10(2):143-50. DOI: 10.1002/cyto.990100205. View

Cook P . The nucleoskeleton and the topology of replication. Cell. 1991; 66(4):627-35. DOI: 10.1016/0092-8674(91)90109-c. View

Banfalvi G, Tanke H, Raap A, Slats J, VAN DER PLOEG M . Early replication signals in nuclei of Chinese hamster ovary cells. Histochemistry. 1990; 94(4):435-40. DOI: 10.1007/BF00266452. View

Zini N, Mazzotti G, Santi P, Rizzoli R, Galanzi A, Rana R . Cytochemical localization of DNA loop attachment sites to the nuclear lamina and to the inner nuclear matrix. Histochemistry. 1989; 91(3):199-204. DOI: 10.1007/BF00490133. View

Carri M, Micheli G, Graziano E, PACE T, BUONGIORNO-NARDELLI M . The relationship between chromosomal origins of replication and the nuclear matrix during the cell cycle. Exp Cell Res. 1986; 164(2):426-36. DOI: 10.1016/0014-4827(86)90041-8. View

Cook P . The nucleoskeleton: artefact, passive framework or active site?. J Cell Sci. 1988; 90 ( Pt 1):1-6. DOI: 10.1242/jcs.90.1.1. View

Basnakian A, Banfalvi G, Sarkar N . Contribution of DNA polymerase delta to DNA replication in permeable CHO cells synchronized in S phase. Nucleic Acids Res. 1989; 17(12):4757-67. PMC: 318030. DOI: 10.1093/nar/17.12.4757. View

Pardoll D, Vogelstein B, Coffey D . A fixed site of DNA replication in eucaryotic cells. Cell. 1980; 19(2):527-36. DOI: 10.1016/0092-8674(80)90527-9. View

Maraldi N, Marinelli F, Cocco L, Papa S, Santi P, Manzoli F . Morphometric analysis and topological organization of nuclear matrix in freeze-fractured electron microscopy. Exp Cell Res. 1986; 163(2):349-62. DOI: 10.1016/0014-4827(86)90066-2. View

10.

Patriotis C, Djondjurov L . Tightly bound DNA-protein complexes representing stable attachment sites of large DNA loops to components of the matrix. Eur J Biochem. 1989; 184(1):157-64. DOI: 10.1111/j.1432-1033.1989.tb15002.x. View

11.

Fox M, Arndt-Jovin D, Jovin T, Baumann P . Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblasts. J Cell Sci. 1991; 99 ( Pt 2):247-53. DOI: 10.1242/jcs.99.2.247. View

12.

Bravo R . Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol. 1987; 105(4):1549-54. PMC: 2114668. DOI: 10.1083/jcb.105.4.1549. View

13.

Berezney R, Buchholtz L . Dynamic association of replicating DNA fragments with the nuclear matrix of regenerating liver. Exp Cell Res. 1981; 132(1):1-13. DOI: 10.1016/0014-4827(81)90076-8. View

14.

van der Velden H, van Willigen G, Wetzels R, Wanka F . Attachment of origins of replication to the nuclear matrix and the chromosomal scaffold. FEBS Lett. 1984; 171(1):13-6. DOI: 10.1016/0014-5793(84)80451-2. View

15.

Mirkovitch J, Mirault M, Laemmli U . Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell. 1984; 39(1):223-32. DOI: 10.1016/0092-8674(84)90208-3. View

16.

Jackson D, Dickinson P, Cook P . Attachment of DNA to the nucleoskeleton of HeLa cells examined using physiological conditions. Nucleic Acids Res. 1990; 18(15):4385-93. PMC: 331255. DOI: 10.1093/nar/18.15.4385. View

17.

Tanaka S . Methods of successive multiparametric cytochemistry and microfluorometry on identical cells with special reference to cell cycle phases in a chick embryo. Exp Cell Res. 1990; 186(1):6-14. DOI: 10.1016/0014-4827(90)90203-m. View

18.

Mazzotti G, Rizzoli R, Galanzi A, Papa S, Vitale M, Falconi M . High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains. J Histochem Cytochem. 1990; 38(1):13-22. DOI: 10.1177/38.1.2403578. View

19.

Verheijen R, Van Venrooij W, Ramaekers F . The nuclear matrix: structure and composition. J Cell Sci. 1988; 90 ( Pt 1):11-36. DOI: 10.1242/jcs.90.1.11. View

20.

Kill I, Bridger J, Campbell K, Maldonado-Codina G, Hutchison C . The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts. J Cell Sci. 1991; 100 ( Pt 4):869-76. DOI: 10.1242/jcs.100.4.869. View