Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus.
We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase delta, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90% of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80% colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.
Somanathan
S
S
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260, USA.
Suchyna
T M
TM
Siegel
A J
AJ
Berezney
R
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D016196:S Phase
D017931:DNA Primers; D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D049452:Green Fluorescent Proteins