S Phase: Synthesis phase •replication of DNA c
Actively dividing eukaryote cells pass through a series of stages knowncollectively as the cell cycle: two gap phases (G1 and G2); anS (for synthesis) phase, in which the genetic material is duplicated; and an Mphase, in which mitosis partitions the genetic material and the cell divides.
of DNA synthesis throughout S phase (Figure 1B; Ray ..
We further examined the localization of Cid-GFP to centromeres when produced at different times in the cell cycle. The deposition of histones onto DNA is predominantly limited to cells that are in S phase of the cell cycle (). We transfected cells with heat shock promoter-histone-GFP fusion constructs (HS-H3GFP or HS-CidGFP). We then induced the construct and pulsed these cells with nucleotide analogues to identify cells in S phase. A pulse of H3-GFP protein localized in early and late replication patterns similar to the nucleotide analogue in the same cells, and no detectable localization occurred in gap-phase cells ( B, left). However, we found that newly produced Cid-GFP protein localized to centromeres in both S-phase and gap-phase cells with similar efficiencies ( B, right). We confirmed that localization of Cid-GFP protein can occur in post-replicative cells, whereas that of H3-GFP is strictly replication dependent, by examining metaphase chromosomes at various times after induction of transfected constructs. In cells transfected with HS-H3GFP, occasional labeled metaphase figures first appear 4 h after induction and are labeled in heterochromatic segments ( A). The pattern of labeling and timing of these figures reflects the deposition of H3-GFP in late S-phase cells and the shortest time required to traverse the G2 phase of the cell cycle, and is consistent with previous observations in Kc cell populations (; ). However, Cid-GFP first appears at the centromeres in 48% of mitotic figures within 2 h of induction: these must be from cells that were induced during the G2 phase ( B). By 4 h s after induction, 100% of the mitotic figures from transfected cells show centromeric labeling. We conclude that the Cid deposition pathway is present and active throughout the cell cycle.
We expect that centromeres must be protected from conventional nucleosome assembly pathways in all dividing cells, but heterochromatin may not always perform this function. For example, distinct heterochromatin does not form in the rapidly dividing nuclei of Drosophila syncytial embryos (), and replication initiates throughout the chromosomes simultaneously (). In these unusual nuclei, conventional nucleosome assembly might be prevented by excluding histone H3 from the apical edge of interphase nuclei, where centromeres lie (). Similarly, varying local concentrations of proteins around nuclei have been proposed to explain progression of the syncytial cell cycle even though bulk cyclin levels are always high (). In later cycles, it appears to be most efficient to produce Cid when centromeres replicate.
Cyclin-dependent kinase 1 - Wikipedia
We wondered whether the deposition of Cid in centromeric replication domains might be facilitated by preventing the deposition of histone H3. Conventional nucleosomes are normally assembled during replication; therefore, we examined the deposition of conventional histones fused to GFP when centromeres replicate. Previous work has shown that histone-GFP fusion proteins can localize to chromatin (; ). We had previously characterized the deposition of H2B-GFP and H3-GFP fusion proteins by examining mitotic chromosomes. When expressed from the cid promoter, which is active early in S phase, these fusion proteins localize to the euchromatic arms of chromosomes (). These experiments did not address whether small quantities of the fusion proteins were also incorporated at centromeres. Therefore, we examined the deposition of histone-GFP fusion proteins after production from transfected cid-promoter constructs in interphase nuclei to determine whether these histones were incorporated in centromeric replication domains. While H2B-GFP was readily deposited at centromeres, H3-GFP was not (P = 0.0002; and ). Both histone-GFP proteins give similar intense labeling in euchromatin, implying that the lack of H3-GFP at centromeres is not due to a general reduced deposition of H3-GFP. Labeling in heterochromatin by H3-GFP and H2B-GFP produced from heat-shock promoter constructs was also indistinguishable in intensity and pattern ( and ). That the centromere may be deficient of histone H3 has been previously suggested, based on the absence of phosphorylated H3 antibody labeling (), and our results with H3-GFP fusion protein support this idea. We conclude that the deposition of H3-containing nucleosomes is inhibited at the time that the cid promoter is active in early S phase.