Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells

Abstract

In eukaryotic cells, prereplication complexes (pre-RCs) are assembled on chromatin in the G1 phase, rendering origins of DNA replication competent to initiate DNA synthesis. When DNA replication commences in S phase, pre-RCs are disassembled, and multiple initiations from the same origin do not occur because new rounds of pre-RC assembly are inhibited. In most experimental organisms, multiple mechanisms that prevent pre-RC assembly have now been identified, and rereplication within the same cell cycle can be induced through defined perturbations of these mechanisms. This review summarizes the diverse array of inhibitory pathways used by different organisms to prevent pre-RC assembly, and focuses on the challenge of understanding how in any one cell type, various mechanisms cooperate to strictly enforce once per cell cycle regulation of DNA replication.The ability of eukaryotic cells to duplicate vast amounts of genetic information quickly and accurately before each cell division is an awe-inspiring product of evolution. To achieve rapid replication, cells use a parallel processing approach in which multiple replisomes copy DNA simultaneously. A dramatic example of this strategy occurs during the early cleavage divisions of the frog Xenopus laevis, in which DNA replication initiates from ∼300,000 sites, called origins, which are spaced ∼10 kb apart (Blow 2001). As a result, 3 billion base pairs of DNA are duplicated within minutes. All eukaryotic organisms use a similar strategy, and the number of origins employed is generally correlated to the genome size and the length of the cell cycle. To ensure that DNA replication is not only fast, but accurate, it is essential that no segment of the chromosome be duplicated more than once. Indeed, reinitiation from even a single origin within the same cell cycle may cause genome instability. Thus, the ability of cells to restrict DNA replication to a single round per cell cycle is a fundamental requirement of cell proliferation and long-term survival. The two-state model for cell cycle regulation of DNA replicationEarly insights into the regulation of eukaryotic DNA replication came from cell fusion experiments (Rao and Johnson 1970), which showed that union of an S-phase cell with a G1 cell accelerates the rate at which the latter enters S phase. In contrast, G2 cells are refractory to this stimulation. These results suggested that the initiation of DNA synthesis requires a positive, diffusible S-phasepromoting activity, and that G1 but not G2-phase cells are competent to respond to this signal. Building on these observations and on their own experiments in Xenopus egg extracts, Blow and Laskey (1988) developed the "licensing" model, in which a licensing factor that is required for replication initiation binds to chromatin in G1. At the G1/S transition, the factor is inactivated, and it cannot be replenished until cells pass through mitosis. These ideas were extended by in vivo footprinting experiments in yeast that showed that origins of replication alternate between two...

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

“…This debate obviously stems from different experimental systems for the detection of endogenous versus 'ectopically' expressed, tagged Cdc6. 7,27,36 Here, we demonstrate that cytoplasmic YFP-tagged Cdc6 binds to chromatin preparations of S phase cells in the same way as endogenous Cdc6, and we provide evidence that this binding takes place during the preparation procedure when the nuclear membrane is lysed by detergents. The fact that the protein remains stably bound to chromatin during subsequent extraction procedures further implies that cytoplasmic Cdc6 retains a high affinity for chromatin.…”

“…In synopsis with the current knowledge about replication licensing and Cdc6 regulation 7,27,36 we suggest the following chronology of regulatory events affecting Cdc6 (Fig. 6): Upon breakdown of the nuclear envelope in prophase, Cdc6 gains access to condensing chromosomes.…”

“…This regulation is mainly achieved by the modulation of specific preRC components via the interplay between cell cycle-specific phosphorylation, dephosphorylation and proteasomal degradation events. 7 For example, loading factor Cdt1 is phosphorylated by Cyclin A/Cdk2 during S phase, which targets it for ubiquitination by E3 ubiquitin ligases and subsequent proteasomal degradation. Its activity is additionally repressed from S to M phase by the presence of the Cdt1 inhibitor geminin.…”

“…[5][6][7] On the one hand, this regulation needs to allow sufficient preRCs to be assembled for a complete replication of the genome. On the other hand, it is as important to prevent further rounds of replication initiated after the beginning of S phase because re-replication of a previously fired origin would lead to the duplication of a chromosome segment and thus to genome instability.…”

Defining replication origin efficiency using DNA fiber assays

“…Phosphorylation of a limited number of initiation factors by CDKs and DDKs triggers the unwinding of the DNA duplex at origins and allows the engagement of the replication machinery (Tanaka et al 2006;Zegerman and Diffley 2006). Importantly, high CDK activity also blocks pre-RC assembly and prevents reinitiation until the next cell cycle (Arias and Walter 2007). Upon initiation, the Mcm2-7 complex acts as an ATPdependent DNA helicase to promote the bidirectional progression of replication forks, together with Cdc45, GINS, and other components of the replisome (Gambus et al 2006).…”