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

Abstract

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.

Atrx deficiency induces telomere dysfunction, endocrine defects, and reduced life span

“…PCNA is a component of the replication machinery and exhibits a distinct staining pattern during S phase. In late S phase, the replication foci are observed in regions of the nucleus containing heterochromatin and as such colocalize with DAPI-bright heterochromatin bundles (38). Atrx-null NPCs accumulated more DNA damage at these late replication foci compared with control cells, confirming an increase in DSBs during the replication process ( Figure 3C).…”

Constitutive Expression of Murine Sak-a Suppresses Cell Growth and Induces Multinucleation

“…Indeed, it was shown that agents which disrupt tubulin assembly and thus inhibit spindle function during mitosis induce nuclear abnormalities in CHO cells (21,30). Correspondingly, we have observed that up to 58% of CHO cells are multinucleated when examined 12 h following their release from a nocodazole block, and these cells are phenotypically indistinguishable from HA-Sak-a-overexpressing cells (5a).…”

Deregulation of cyclin E in human cells interferes with prereplication complex assembly

“…Therefore, we analyzed the localization patterns of BrdU and proliferating cell nuclear antigen (PCNA) in individual early S phase cells with or without constitutive cyclin E expression. As a processivity factor for DNA polymerase δ during replication ( Prelich et al, 1987 ; Krishna et al, 1994 ; Fukuda et al, 1995 ; Kelman, 1997 ), PCNA has been shown to be localized at the replication fork and to colocalize with newly synthesized DNA labeled with BrdU ( Hozak et al, 1993 ; Takanari et al, 1994 ; Somanathan et al, 2001 ). Therefore, we used PCNA as a quantitative marker for replication forks.…”