Replication sites as revealed by double label immunofluorescence against proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) in synchronized CHO cells and vincristine-induced multinucleate cells.

Double label immunofluorescence against PCNA and BrdU clearly revealed several characteristics of DNA replication sites in synchronized CHO cells. We observed that the distribution of replication sites changed from early to late S phase, particularly in the nucleolar and perinuclear regions, and the amount of PCNA in each replication site markedly decreased or disappeared with the progression of S phase. Although co-localization of PCNA and BrdU was usually seen, the intensity of fluorescence occasionally differed between the labeled PCNA and BrdU, particularly in late S phase. Based on the assumption that such a difference may reflect a different configuration of the chromatin, we propose that the brightly granular fluorescent staining of PCNA or BrdU in early S phase indicates a condensed segment of euchromatin. In the late S phase nucleus, we clearly observed a chromatin-like structure in the late replicating segments of heterochromatin in anti-PCNA stained material. In vincristine-induced multinucleate cells, a discrepancy between PCNA-distribution and BrdU-incorporation in sister nuclei was sometimes seen. Such observations indirectly support the following two mechanisms for premature chromosome condensation proposed by others: asynchronous initiation of DNA synthesis; and a difference in the rate of DNA synthesis. In addition, the finding that BrdU was not incorporated into sites that had PCNA deposits suggests a third mechanism: the local disturbance of replication sites. Furthermore, unusual distribution patterns for replication sites suggest that the nucleation process in multinucleate cells differs from that in normal cells.