Dynamics of replication foci and nuclear matrix during S phase in Allium cepa L. cells

Abstract

The sequential organisation of replication foci during S phase in onion ( Allium cepa) and their relationship to the nuclear matrix were investigated. To discern their structural features and temporal firing sequence, immunodetection of 5-bromo-2'-deoxyuridine (BrdU) was carried out after in vivo feeding in synchronised cells released from a 14-h-long hydroxyurea block. Replication foci consisted of small replication granules, called replisomes, which clustered together. Analysis of synchronous binucleate cells that maintained in their two nuclei the specular symmetry of distribution of sister chromosomes in anaphase, showed that replication starts in small replication foci at the telomeric pole (pattern I), though the telomeres themselves formed large foci that were late-replicating. The rDNA replication foci (pattern II) also become replicated in early S phase. Replication of large foci, including the heterochromatin (IV), occurred in late S phase and finished at the centromeric nuclear pole (pattern V). Labelling of proliferating cell nuclear antigen (PCNA) in nuclear matrices, prepared from S-phase nuclei after extensive DNase digestion, demonstrated that replication foci were always stably anchored to the nuclear matrix. Thus, association with the nucleoskeleton is not exclusively mediated by the replicating or nascent DNA. The overlapping of patterns I, II and III in the nuclear matrix, in contrast to the results of BrdU localisation in nuclei, suggests that PCNA becomes associated with the nuclear matrix before the replication foci are operative, and remains bound during replication.

Visualizing the dynamics of chromosome structure formation coupled with DNA replication

“…Numbers of supporting examples have actually showed the relationship of DNA replication and chromosome conformation (Zink et al 1998;Manders et al 1999;Samaniego et al 2002). However, the results shown in these reports were derived from the observation in interphase nuclei; thus, the resolution and the resulting information were limited because chromosomes are only visible in the mitotic stage of the cell cycle and are invisible as decondensed structure in interphase (Manders et al 1999;Gotoh and Durante 2006).…”

“…Much recently accumulated evidences have implied that eukaryote DNA replication/ transcription is involved in the compaction of chromosomes (Zink et al 1998;Manders et al 1999;Samaniego et al 2002); however, its involvement remains unclear.…”

Host DNA Replication Is Induced by Geminivirus Infection of Differentiated Plant Cells

“…The punctate labeling of host DNA after a 4-h treatment and the uniform incorporation of BrdU into condensed chromatin are not consistent with DNA repair. Punctate BrdU labeling of host DNA has been reported to be associated with DNA replication factories in onion cells (Samaniego et al, 2002) and animal cells (Cossmann et al, 2000). These results establish TGMV infection of N. benthamiana plants as a useful system for analyzing the molecular basis for how plants control DNA replication and demonstrate that host DNA replication can be caused by a plant virus.…”

“…One possibility is that early in infection, AL1 forms protein complexes that bind to pRBR, inducing cells to reenter the cell cycle, but do not support efficient initiation of viral DNA synthesis (Settlage et al, 2001). Nonetheless, host DNA replication may proceed as induced host DNA replication factors, such as PCNA, are integrated at chromosomal replication initiation complexes (Samaniego et al, 2002). Later in infection, AL1 forms replication-competent oligomers and may "compete" with host DNA to recruit the available replication factors.…”

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

“…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).…”