The functional role of Cdc6 in S-G2/M in mammalian cells

The Cdc6 protein is required for licensing of replication origins before the onset of DNA replication in eukaryotic cells. Here, we examined whether Cdc6 has other roles in mammalian cell-cycle progression from S to G2/M phase. Using RNA interference, we showed that depletion of Cdc6 in synchronous G1 cells blocks G1 to S transition, confirming the essential role of Cdc6 in the initiation of DNA replication. In contrast, depletion of Cdc6 in synchronous S-phase cells slowed DNA replication and led to mitotic lethality. The Cdc6-depleted S-phase cells showed fewer newly fired origins; however, established replication forks remained active, even during chromatin condensation. Despite such DNA replication abnormalities, loss of Cdc6 failed to activate Chk1 kinase. These results show that Cdc6 is not only required for G1 origin licensing, but is also crucial for proper S-phase DNA replication that is essential for DNA segregation during mitosis.     

LRR1-mediated replisome disassembly promotes DNA replication by recycling replisome components

After two converging DNA replication forks meet, active replisomes are disassembled and unloaded from chromatin. A key process in replisome disassembly is the unloading of CMG helicases (CDC45–MCM–GINS), which is initiated in Caenorhabditis elegans and Xenopus laevis by the E3 ubiquitin ligase CRL2^LRR1. Here, we show that human cells lacking LRR1 fail to unload CMG helicases and accumulate increasing amounts of chromatin-bound replisome components as cells progress through S phase. Markedly, we demonstrate that the failure to disassemble replisomes reduces the rate of DNA replication increasingly throughout S phase by sequestering rate-limiting replisome components on chromatin and blocking their recycling. Continued binding of CMG helicases to chromatin during G2 phase blocks mitosis by activating an ATR-mediated G2/M checkpoint. Finally, we provide evidence that LRR1 is an essential gene for human cell division, suggesting that CRL2^LRR1 enzyme activity is required for the proliferation of cancer cells and is thus a potential target for cancer therapy.     

Global regulation of genome duplication in eukaryotes: an overview from the epifluorescence microscope

In eukaryotes, DNA replication is initiated along each chromosome at multiple sites called replication origins. Locally, each replication origin is “licensed” or specified at the end of the M and the beginning of the G1 phases of the cell cycle. During the S phase when DNA synthesis takes place, origins are activated in stages corresponding to early and late-replicating domains. The staged and progressive activation of replication origins reflects the need to maintain a strict balance between the number of active replication forks and the rate at which DNA synthesis proceeds. This suggests that origin densities (frequency of initiation) and replication fork movement (rates of elongation) must be coregulated to guarantee the efficient and complete duplication of each subchromosomal domain. Emerging evidence supports this proposal and suggests that the ATM/ATR intra-S phase checkpoint plays an important role in the coregulation of initiation frequencies and rates of elongation. In this paper, we review recent results concerning the mechanisms governing the global regulation of DNA replication and discuss the roles these mechanisms play in maintaining genome stability during both a normal and perturbed S phase.     

Independent positioning and action of Escherichia coli replisomes in live cells

A prevalent view of DNA replication has been that it is carried out in fixed "replication factories." By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.     

A Yeast GSK-3 Kinase Mck1 Promotes Cdc6 Degradation to Inhibit DNA Re-Replication

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCF^CDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.     

Chromatin structure restricts origin utilization when quiescent cells re-enter the cell cycle

Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. Quiescent cells contained very low levels of replication initiation factors, and their entry into S phase was delayed until these factors were re-synthesized. A longer S phase in these cells correlated with the activation of fewer origins of replication compared to G1 cells. The chromatin structure around inactive origins in G0 cells showed increased H3 occupancy and decreased nucleosome positioning compared to the same origins in G1 cells, inhibiting the origin binding of the Mcm4 subunit of the MCM licensing factor. Thus, quiescent yeast cells are under-licensed during their re-entry into S phase.     

The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.

The budding yeast Cdc6 protein (Cdc6p) is essential for formation of pre-replicative complexes (pre-RCs) at origins of DNA replication. Regulation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle. Cdc6p is normally only present at high levels during the G1 phase of the cell cycle. This is partly because the CDC6 gene is only transcribed during G1. In this article we show that rapid degradation of Cdc6p also contributes to this periodicity. Cdc6p degradation rates are regulated during the cell cycle, reaching a peak during late G1/early S phase. Removal of a 47-amino-acid domain near the N-terminus of Cdc6p prevents degradation of Cdc6p. Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-mediated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p. A stable Cdc6p mutant which is no longer degraded by the Cdc4/34/53 pathway is, none the less, fully functional. Constitutive overexpression of either wild-type or stable Cdc6p does not induce re-replication and does not induce assembly of pre-replicative complexes after DNA replication is complete.     

Uncoupling of sister replisomes during eukaryotic DNA replication

The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes. Some data imply that they travel away from one another and thus function independently. Alternatively, sister replisomes may form a stationary, functional unit that draws parental DNA toward itself. If this "double replisome" model is correct, a constrained DNA molecule should not undergo replication. To test this prediction, lambda DNA was stretched and immobilized at both ends within a microfluidic flow cell. Upon exposure to Xenopus egg extracts, this DNA underwent extensive replication by a single pair of diverging replisomes. The data show that there is no obligatory coupling between sister replisomes and, together with other studies, imply that genome duplication involves autonomously functioning replisomes.     

The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells

The initiation of DNA synthesis is governed by the licensing of replication origins, which consists of assembling a pre-replication complex (pre-RC) on origins during late M- and G1-phases. In metazoans, functional replication origins do not show defined DNA consensus sequences, thus evoking the involvement of chromatin determinants in the selection of these origins. Here, we show that the onset of licensing in mammalian cells coincides with an increase in histone H4 Lys 20 monomethylation (H4K20me1) at replication origins by the methyltransferase PR-Set7 (also known as Set8 or KMT5A). Indeed, tethering PR-Set7 methylase activity to a specific genomic locus promotes the loading of pre-RC proteins on chromatin. In addition, we demonstrate that PR-Set7 undergoes a PCNA- and Cul4-Ddb1-driven degradation during S phase that contributes to the disappearance of H4K20me1 at origins and the inhibition of replication licensing. Strikingly, expression of a PR-Set7 mutant insensitive to this degradation causes the maintenance of H4K20me1 and repeated DNA replication at origins. These results elucidate a critical role for PR-Set7 and H4K20me1 in the chromatin events that regulate replication origins.     

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

To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.     

Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.     

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks

Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome‐wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild‐type Rif1 and truncated Rif1 lacking the Rap1‐interaction domain, we identify hundreds of Rap1‐dependent and Rap1‐independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1‐independent manner, associating with both early and late‐initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA. Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks.     

CDK-Dependent Stabilization of Cdc6: Linking Growth and Stress Signals to Activation of DNA Replication

Cyclin-dependent kinases (CDKs) play a crucial role in cell cycle progression by controlling the transition from G1 phase into S phase where DNA is replicated. Key to this transition is the regulation of initiation of DNA replication at replication origins. CDKs are thought to regulate origins of replication both positively and negatively by phosphorylating replication proteins at origins. Several replication proteins that are potentially negatively regulated upon CDK phosphorylation have been identified. However, the mechanism by which CDKs activate replication is currently less well understood. New observations revealing that the initiation protein Cdc6 is stabilized by CDK2-dependent phosphorylation may give more insight in this process.     

Cdt1 Interactions in the Licensing Process: A Model for Dynamic Spatio-temporal Control of Licensing

Within each cell cycle, a cell must ensure that the processes of selection of replication origins (licensing) and initiation of DNA replication are well coordinated to prevent re-initiation of DNA replication from the same DNA segment during the same cell cycle. This is achieved by restricting the licensing process to G1 phase when the prereplicative complexes (preRCs) are assembled onto the origin DNA, while DNA replication is initiated only during S phase when de novo preRC assembly is blocked. Cdt1 is an important member of the preRC complex and its tight regulation through ubiquitin-dependent proteolysis and binding to its inhibitor Geminin ensure that Cdt1 will only be present in G1 phase, preventing relicensing of replication origins. We have recently reported that Cdt1 associates with chromatin in a dynamic way and recruits its inhibitor Geminin onto chromatin in vivo. Here we discuss how these dynamic Cdt1-chromatin interactions and the local recruitment of Geminin onto origins of replication by Cdt1 may provide a tight control of the licensing process in time and in space.     

DDK promotes DNA replication initiation: Mechanistic and structural insights

DNA replication initiation in eukaryotes is tightly regulated through two cell-cycle specific processes, replication licensing to install inactive minichromosome maintenance (MCM) double-hexamers (DH) on origins in early G1 phase and origin firing to assemble and activate Cdc45-Mcm2-7-GINS (CMG) helicases upon S phase entry. Two kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are responsible for driving the association of replication factors with the MCM-DH to form CMG helicases for origin melting and DNA unwinding and eventually replisomes for bi-directional DNA synthesis. In recent years, cryo-electron microscopy studies have generated a collection of structural snapshots for the stepwise assembly and remodeling of the replication initiation machineries, creating a framework for understanding the regulation of this fundamental process at a molecular level. Very recent progress is the structural characterization of the elusive MCM-DH-DDK complex, which provides insights into mechanisms of kinase activation, substrate recognition and selection, as well as molecular role of DDK-mediated MCM-DH phosphorylation in helicase activation.     

Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ～10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3-related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.     

Initiation of DNA replication at a nuclear matrix-attached chromatin fraction

It is still unclear what nuclear components support initiation of DNA replication. To address this issue, we developed a cell-free replication system in which the nuclear matrix along with the residual matrix-attached chromatin was used as a substrate for DNA replication. We found out that initiation occurred at late G1 residual chromatin but not at early G1 chromatin and depended on cytosolic and nuclear factors present in S phase cells but not in G1 cells. Initiation of DNA replication occurred at discrete replication foci in a pattern typical for early S phase. To prove that the observed initiation takes place at legitimate DNA replication origins, the in vitro synthesized nascent DNA strands were isolated and analyzed. It was shown that they were enriched in sequences from the core origin region of the early firing, dihydrofolate reductase origin of replication ori-beta and not in distal to the origin sequences. A conclusion is drawn that initiation of DNA replication occurs at discrete sub-chromosomal structures attached to the nuclear matrix.     

Replication origins: why do we need so many?

During the G1 phase of the cell cycle, replication origins are prepared to fire, a process that is referred to as origin licensing. It was often pondered what a cell's fate would be if not all of its replication origins were licensed and subsequently activated during S phase. One obvious prediction was that S phase would simply be prolonged. As it turns out, however, the consequences are much more complex. A short G1 phase enforced by premature entry into S phase, or other events that negatively affect origin licensing, will ultimately compromise the cell's ability to complete DNA replication before entering mitosis. As a result, the cell becomes genomically unstable when it attempts to repair unreplicated DNA during anaphase. Thus, the density of active replication origins in the chromosomes of eukaryotic cells determines S phase dynamics and chromosome stability during mitosis.     

Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex.

The transition from G1 to S phase of the cell cycle may be regulated by modification of proteins which are essential for initiating DNA replication. One of the first events during initiation is to unwind the origin DNA and this requires a single-stranded DNA binding protein. RPA, a highly conserved multi-subunit single-stranded DNA binding protein, was first identified as a cellular protein necessary for the initiation of SV40 DNA replication. The 32 kDa subunit of RPA has been shown to be phosphorylated at the start of S phase. Using SV40 replication as a model, we have reproduced in vitro the S phase-dependent phosphorylation of RPA-32 and show that it occurs specifically within the replication initiation complex. Phosphorylated RPA-32 is predominantly associated with DNA. Phosphorylation is not a pre-requisite for association with DNA, but occurs after RPA binds to single-stranded DNA formed at the origin during the initiation phase. The protein kinase(s) which phosphorylates RPA-32 is present at all stages of the cell cycle but RPA-32 does not bind to the SV40 origin or become phosphorylated in extracts from G1 cells. Therefore, the cell cycle-dependent phosphorylation of RPA-32 may be regulated by its binding to single-stranded origin DNA during replication initiation.