Dynamics of DNA Replication Factories in Living Cells

doi:10.1083/jcb.149.2.271

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© The Rockefeller University Press, 0021-9525/2000/4/271/ $5.00
The Journal of Cell Biology, Volume 149, Number 2, April 17, 2000 271-280

Original Article

Dynamics of DNA Replication Factories in Living Cells

Heinrich Leonhardt^a, Hans-Peter Rahn^a, Peter Weinzierl^,b, Anje Sporbert^a, Thomas Cremer^b, Daniele Zink^b, and M. Cristina Cardoso^a
^a Max Delbrück Center for Molecular Medicine, D-13125 Berlin, Germany
^b Institute for Anthropology and Human Genetics, LMU, D-80336 Munich, Germany

Correspondence to: M. Cristina Cardoso, Franz Volhard Clinic, Wiltbergstr. 50, 13125 Berlin, Germany. Tel:49-30-9417-2341 Fax:49-30-9417-2336 E-mail:cardoso{at}fvk-berlin.de.

DNA replication occurs in microscopically visible complexesat discrete sites (replication foci) in the nucleus. These fociconsist of DNA associated with replication machineries, i.e.,large protein complexes involved in DNA replication. To studythe dynamics of these nuclear replication foci in living cells,we fused proliferating cell nuclear antigen (PCNA), a centralcomponent of the replication machinery, with the green fluorescentprotein (GFP). Imaging of stable cell lines expressing low levelsof GFP-PCNA showed that replication foci are heterogeneous insize and lifetime. Time-lapse studies revealed that replicationfoci clearly differ from nuclear speckles and coiled bodiesas they neither show directional movements, nor do they seemto merge or divide. These four dimensional analyses suggestedthat replication factories are stably anchored in the nucleusand that changes in the pattern occur through gradual, coordinated,but asynchronous, assembly and disassembly throughout S phase.

Key Words: cell cycle, DNA replication foci, green fluorescent protein,nuclear organization, proliferating cell nuclear antigen

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