Clusters, factories and domains: The complex structure of S-phase comes into focus

Abstract

During S-phase of the cell cycle, chromosomal DNA is replicated according to a complex replication timing program, with megabase-sized domains replicating at different times. DNA fibre analysis reveals that clusters of adjacent replication origins fire near-synchronously. Analysis of replicating cells by light microscopy shows that DNA synthesis occurs in discrete foci or factories. The relationship between timing domains, origin clusters and replication foci is currently unclear. Recent work, using a hybrid Xenopus/hamster replication system, has shown that when CDK levels are manipulated during S-phase the activation of replication factories can be uncoupled from progression through the replication timing program. Here, we use data from this hybrid system to investigate potential relationships between timing domains, origin clusters and replication foci. We suggest that each timing domain typically comprises several replicon clusters, which are usually processed sequentially by replication factories. We discuss how replication might be regulated at different levels to create this complex organisation and the potential involvement of CDKs in this process.

Figure 1

The replication timing program. (A) CHO cells, previously synchronized at the G₁/S border and released into S-phase for various times, were pulse labelled with BrdU for 5 min and stained with anti-BrdU antibodies to visualize patterns of DNA replication. Shown are characteristic examples of each of the 5 labeling patterns (Types I–V). Reproduced from Dimitrova & Gilbert 1999. (B) The replication cycle—the establishment and execution of replication timing in the cell cycle. The innermost wheel marks the phases of the cell cycle and the outermost wheel the key stages of DNA replication, where the periods of licensing competence and inhibition are shown in yellow and red respectively and the execution of the replication timing program during S-phase is shown in green. Key nuclear transitions are represented diagrammatically in the central portion. During mitosis, paired sister chromatids aligned on the metaphase plate are separated to opposite poles of the dividing cells upon entry into anaphase. Late mitotic chromosome decondensation then readies DNA for emergence into G₁. In early G₁, after nuclear envelope reformation (continuous black line), regions of euchromatin, shown in red and heterochromatin, shown in blue, initially randomly dispersed, move to occupy specific domains within the nucleus. During S-phase, replication foci, shown in green, are activated in accordance with the execution of the timing program. Post-replicative nuclei pass through G₂ into mitosis, where chromosome condensation and nuclear envelope breakdown (dashed black line) ready the cell for sister chromatid separation and cell division. The execution of the timing decision point and the origin decision point are marked.

Figure 2

Progression of CHO nuclei through the replication timing program in Xenopus egg extract. CHO nuclei, incubated in Xenopus egg extracts, were pulse labelled for 5 min, at various times, with Cy3-dUTP to directly visualize patterns of DNA replication. Shown are characteristic examples of each of the 9 labeling patterns, the 5 distinct patterns (Types I–V) and the 4 overlapping patterns (Types I/II, II/III, III/IV, IV/V). Reproduced from Thomson et al., 2010.

Figure 3

Decoupling of DNA replication and the replication timing program. CHO nuclei were incubated in Xenopus egg extracts ±10 µM roscovitine (A) or 1 pM cyclin A (B). At various times, aliquots were pulse labelled with Cy3-dUTP to assess the proportion of different replication patterns. At the same time DNA synthesis was measured in extracts supplemented with α-[³²P]dATP by TCA precipitation and scintillation counting. The replication pattern at different times is plotted against total DNA synthesis. Reproduced from Thomson et al., 2010.

Figure 4

Activation of replication foci depends on CDK levels. (A) CHO nuclei, were incubated in Xenopus egg extracts for 50 min ± 10 µM roscovitine or 3 pM cyclin A and then pulse labelled for 5 min with Cy3-dUTP (red) and then isolated, stained with DAPI (blue) and visualized. Representative images for each condition are shown. Reproduced from Thomson et al., 2010. (B) CDK sensitivity of DNA replication control. Cartoon showing three different levels of S phase control. The upper level shows progression between two different stages of the timing program for a single nucleus, where the small green dots represent replication factories and the black circle the nuclear envelope. The middle level represents the firing of replication origins (pink dots) in a newly activated replication factory next to an existing factory (large green dot). The lower level shows initiation of a new replication origin on a strand of DNA in an active replicon cluster within a replication factory, where the pink circle represents the new origin and the green line the DNA.

Figure 5

The organization of replicon clusters. (A) CHO nuclei were incubated in Xenopus egg extracts supplemented with α-[³²P]dATP. At 40 min an aliquot was supplemented with 1 mM roscovitine to block further initiation events. At various times throughout the length of the incubation total DNA synthesis was measured by TCA precipitation and scintillation counting. Reproduced from Thomson et al., 2010. (B) Replication fork fusion within a replicon cluster reduces replication rate. Cartoon depicting the effect of replication fork fusion on reducing replication rate within a replicon cluster, where DNA is represented by a black line and open figures represent ‘replication bubbles’, the ongoing synthesis of DNA from a pair of replication forks moving in opposite directions. Replication fork number and density and replication fork rate and termination time, within a cluster, are indicated.

Figure 6

Replication timing domains are comprised of multiple replicon clusters. (A) Genomic replication timing profile of 158.4–162.7 Mbp of mouse chromosome 2, in neural precursor (NPC/ASd6) cells., A broad region of DNA with approximately coordinately timed replication initiation constitutes a replication timing domain. Within the fine detail of each timing domain smaller divisions of replication timing, perhaps representing individual replicon clusters, can be discerned. Data prepared using www.replicationdomain.org. (B and C) Cartoon showing hypothetical replicon cluster activation within a replication timing domain similar to the one shown in (A), in (B) CHO cells and (C) the hybrid system. Currently active clusters, those in which the internal forks are still active and have not yet fused, are depicted in red. Completed replicon clusters, those in which the internal forks have fused but the outermost forks remain active, are depicted in blue. Unreplicated DNA is represented by the black line. The size of a cluster in the 2 systems is scaled relative to their observed length.

Figure 7

Different models for how the replication timing program might operate. Groups of replication origins, represented as black circles, on early-, mid- and late-replicating DNA are shown in each panel. CDKs acting on these origins to promote initiation and turn them into replication bubbles, are depicted as red vertical arrows. (A) Initiation of origins in one timing stage is dependent on initiation of the previous stage having been completed. (B) Initiation of later origins requires increased CDK activity, so the replication timing program is driven by rising CDK levels. (C) CDK activity is required not only to promote origin activation (vertical arrows), but also to drive the replication timing program (horizontal arrows), so that later origins become competent for initiation.