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 compu ter image analysis. Labeling (5 min pulse) of DNA replication sites in norm al human diploid fibroblast cells (NHF1) with BrdU was followed by immunost aining with PCNA antibodies. A striking degree of colocalization was seen b etween PCNA and the characteristic patterns of DNA replication sites of ear ly, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 10 8:1-11). These observations were confirmed by quantitative computer image a nalysis which revealed that approximately 90% of the PCNA-stained area over lapped with DNA replication sites in early S-phase. Pulse-chase experiments , involving in vivo labeling for replication followed by PCNA staining at l ater time points, suggested that PCNA disassembles from previously replicat ed sites and targets to newly active sites of DNA replication. To further s tudy this phenomenon in living cells, stable GFP-PCNA transfectants under t he control of a tetracycline-inducible promoter were created in mouse 3T6 c ells. 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 r evealed progressive changes in the GFP-PCNA distribution that mimic the rep lication patterns observed in fixed cells. We conclude that GFP-PCNA target s to DNA replication sites in living cells and is an effective marker for t racking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase. (C) 2001 Wiley-Liss. Inc.