The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells

Abstract

We investigated the organization of DNA replication sites in primary (young or presenescent), immortalized and transformed mammalian cells. Four different methods were used to visualize replication sites: in vivo pulse-labeling with 5-bromo-2′-deoxyuridine (BrdU), followed by either acid depurination, or incubation in nuclease cocktail to expose single-stranded BrdU-substituted DNA regions for immunolabeling; biotin-dUTP labeling of nascent DNA by run-on replication within intact nuclei and staining with fluorescent streptavidin;and, finally, immunolabeling of the replication fork proteins PCNA and RPA. All methods produced identical results, demonstrating no fundamental differences in the spatio-temporal organization of replication patterns between primary, immortal or transformed mammalian cells. In addition, we did not detect a spatial coincidence between the early firing replicons and nuclear lamin proteins, the retinoblastoma protein or the nucleolus in primary human and rodent cells. The retinoblastoma protein does not colocalize in vivo with members of the Mcm family of proteins (Mcm2, 3 and 7) at any point of the cell cycle and neither in the chromatin-bound nor in the soluble nucleoplasmic fraction. These results argue against a direct role for the retinoblastoma or nuclear lamin proteins in mammalian DNA synthesis under normal physiological conditions.

Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells

“…DNA replication shows a global level of spatiotemporal patterning throughout S phase that can be categorized into early, mid, and late S phase patterns based on the distribution of DNA replication foci (31,34,70,72,73). Replication foci are discrete sub-nuclear sites where multiprotein complexes involved in DNA replication assemble at replication forks (91).…”

cAMP-mediated Inhibition of DNA Replication and S Phase Progression: Involvement of Rb, p21Cip1, and PCNA

“…Furthermore, after irradiation of S phase-synchronized cells, hypophosphorylated Rb has been reported to be directed to certain chromosomal replication initiation sites, at or after the time they had begun to fire, as part of the process whereby Rb suppresses abnormal rereplication activity (Avni et al, 2003). Although the interaction of Rb with proteins at the replication origins or its presence at the replication foci has been disputed (Dimitrova and Berezney, 2002;Angus et al, 2004), we cannot exclude the possibility that after dephosphorylation, Rb might be recruited to replication initiation sites at or after the time they have begun to fire. This might, in turn, control any series of steps in initiation such as origin recognition, DNA unwinding, or it can block the switching from pol ␣-dependent primer formation to processive replication using pol ␦ and PCNA, and thus lead to inhibition of DNA replication.…”

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips

“…These differences in chromosome organization should be considered together with other larger scale differences in the spatial organization of replication within the nucleus. In maize, mid S replicating loci do not cluster around the nuclear and nucleolar periphery (Bass et al, 2015) as they do in the mammalian nucleus (Dimitrova and Berezney, 2002;Panning and Gilbert, 2005;Zink, 2006). Instead, numerous foci are distributed throughout the nucleoplasm during both early and mid S phase (Bass et al, 2015).…”

“…Some information is available about mid replicating chromatin from cytological studies. For example, in mammalian cell lines, mid replicating loci are spatially localized to the perinuclear and perinucleolar edges, while early replication occurs in dispersed, punctate foci (Dimitrova and Berezney, 2002;Panning and Gilbert, 2005;Zink, 2006). Such spatiotemporal differences are less pronounced in plants, in which both early and mid replicating loci have been reported to be dispersed throughout the nucleoplasm (Samaniego et al, 2002;Bass et al, 2015).…”