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

Dynamics of DNA Replication Factories in Living Cells

^a Max Delbrück Center for Molecular Medicine, D-13125 Berlin, Germany
^b Institute for Anthropology and Human Genetics, LMU, D-80336 Munich, Germany
Franz Volhard Clinic, Wiltbergstr. 50, 13125 Berlin, Germany.49-30-9417-233649-30-9417-2341

cardoso{at}fvk-berlin.de

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and 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

© 2000 The Rockefeller University Press

Abbreviations used in this paper: 4D, four-dimensional; BrdU, 5-bromo-2'-deoxyuridine; GFP, green fluorescent protein; PCNA, proliferating cell nuclear antigen.

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