Dynamics of DNA replication factories in living cells.
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.
Leonhardt
H
H
Max Delbrück Center for Molecular Medicine, D-13125 Berlin, Germany.
Rahn
H P
HP
Weinzierl
P
P
Sporbert
A
A
Cremer
T
T
Zink
D
D
Cardoso
M C
MC
D000818:Animals; D019556:COS Cells; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology* / Q000648:ultrastructure; D004261:DNA Replication; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D018715:Microscopy, Video / Q000379:methods; D018482:Muscle, Skeletal; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins