However, it is still possible that they have overlapping functions, as the effect of depleting both Dbf4 and Drf1 has not been examined. An abundance of genetic and biochemical data indicates that the Mcm2—7 complex is the physiological substrate for DDK required for replication initiation. Multiple subunits of the Mcm2—7 complex have been shown to be phosphorylated by DDK in vitro and in vivo Lei et al.
A point mutation in a highly conserved proline in Mcm5 to leucine bypasses the need for DDK activity for replication in S. Genomic footprinting of this Mcm5 mutant revealed structural changes at origins that resemble those that occur in S phase after Cdc7 activation, suggesting that the Mcm5 mutant was prematurely activated, thereby forgoing the need for DDK Geraghty et al. When the structure of the archaeal homologue of Mcm2—7 was solved, the conserved proline was found to be buried in a region between two closely packed domains Fletcher et al.
Therefore, replacing the proline with a bulky side chain is predicted to alter the structure of the protein by pushing apart the two domains. Consistent with this hypothesis, mutating the proline of S. The latter model is attractive because it implies that replication initiation, specifically the loading of Cdc45, requires two independent signals: a replication signal involving pre-RC formation and DDK activity, and a cell cycle signal propagated through CDK activity.
The final step in replication initiation is the loading of the replicative polymerases reviewed in Waga and Stillman, ; Kawasaki and Sugino, ; Hubscher et al. Although all three polymerases share a conserved catalytic core, they perform specialized functions in elongation. Since the C-terminus is involved in checkpoint control Navas et al. During replication, histones are divided among the parent and daughter strands and new histones are assembled by chromatin assembly factors CAFs that travel with the replication fork Shibahara and Stillman, ; Zhang et al.
How the histone modifications, which contain information for regulating gene expression, are then copied onto the newly assembled histones on DNA is an intense area of current research for recent reviews, see McNairn and Gilbert, ; Ehrenhofer-Murray, and references therein.
Until this point we have discussed the ordered series of events that lead to origin firing during S phase. But how does a cell ensure that each origin fires only once per cell cycle? Repeated rounds of initiation result in re-replicated DNA, leading to chromosomal breakage and genomic instability. Clues to our understanding of how this is accomplished came from early cell fusion experiments Rao and Johnson, When a G1 cell is fused to an S phase cell, the G1 nucleus begins to replicate prematurely.
Therefore, a G1 nucleus is competent to replicate, but lacks factors present in S phase that are necessary for origin firing. However, delivering these factors to a G2 nucleus by fusing an S and G2 cell fails to initiate replication in the G2 nucleus, indicating that the cell has some means of differentiating between replicated and unreplicated DNA.
These observations led to the concept of replication licensing Blow and Hodgson, ; Nishitani and Lygerou, The license is then restored after cell division. By restricting replication licensing to G1, each round of replication must have an intervening cell division, ensuring that DNA is replicated only once per cell cycle. What is the nature of this license? It has now been established that loading of the Mcm2—7 complex represents the license for replication Blow and Hodgson, ; Nishitani and Lygerou, Therefore, regulation of pre-RC formation is critical for limiting replication to once per cell cycle Table 1.
The primary regulator of replication licensing is CDK. The increase in CDK activity at the transition to S phase has two important functions for regulating pre-RC formation and function. Firstly, CDK activates factors necessary for origin firing, which concomitantly disassembles the pre-RC, leaving behind an unlicensed origin. Therefore, CDK is responsible for both removing the license and preventing relicensing in S phase.
High CDK activity persists until the M—G1 transition, at which time cyclins are rapidly degraded and origins are again competent to be licensed. Consistent with this model, inhibition of CDK activity in G2 is sufficient to stimulate rereplication Broek et al. CDK inhibition prior to mitosis is also the mechanism responsible for the physiological rereplication that occurs during certain stages of Drosophila development Follette et al.
CDK phosphorylation exerts its inhibitory effect on pre-RC formation by negatively regulating the action of licensing factors. Each component is controlled independently by CDKs providing redundant pathways that protect against rereplication and genomic instability Nguyen et al. The most well-studied factor regulated by CDK is Cdc6, which is exported from the nucleus following CDK phosphorylation in vertebrate cells Saha et al.
In mammalian cells, a significant fraction of Cdc6 also remains associated with chromatin throughout the cell cycle Coverley et al. Phosphorylation of chromatin-bound Cdc6 has no effect on its localization, but inactivates it in some manner Coverley et al.
Cdt1 phosphorylation by CDK also targets it for degradation during S phase in mammalian cells Liu et al. The Mcm2—7 complex travels with the replication fork and hence are displaced from origins upon initiation Aparicio et al. Mcms that are released from chromatin are exported from the nucleus in S.
Mcms remain nuclear throughout the cell cycle in other eukaryotes, but evidence suggest that CDK phosphorylation decreases the affinity of Mcms for chromatin Coue et al. In mammalian cells, Orc1's affinity for chromatin diminishes after origin firing during S phase, at which time it is monoubiquitinated in hamster cells Li and DePamphilis, or polyubiquitinated and degraded by the proteasome in mammalian cells Mendez et al. Orc2—5 levels remain stable and chromatin bound throughout the cell cycle; however, that does not preclude the possibility that CDK phosphorylation regulates the function of other ORC subunits.
In yeast, the entire ORC complex remains bound to origins throughout the cell cycle. The discovery that ORC phosphorylation is involved in preventing rereplication in S.
Preventing CDK recruitment or mutating the CDK phosphorylation sites in Orc2 and Orc6 contributes to rereplication, indicating their role in preventing relicensing Nguyen et al.
In a similar fashion, S. Disrupting this interaction results in relicensing of origins after S phase and complete genomic reduplication Wuarin et al. It remains to be determined how phosphorylation of ORC abrogates its function. Multicellular eukaryotes have evolved a second mechanism to prevent relicensing and rereplication involving the protein Geminin. Geminin forms a dimer that inhibits licensing by directly interacting with Cdt1 McGarry and Kirschner, ; Wohlschlegel et al.
Although Geminin forms a complex with Cdt1 in solution Wohlschlegel et al. Geminin is loaded onto chromatin as cells enter S phase at about the same time as Cdc45 and persists on chromatin after Cdt1 leaves Maiorano et al. Recently described structure—function studies suggest that Geminin and Cdt1 execute a bipartite interaction, and confirms biochemical data that Geminin binding prevents Cdt1 from interacting with Mcms by steric hindrance Lee et al.
Regulation of Geminin in somatic cells resembles CDK activity, being low in G1 and accumulating during S and G2, after which it is ubiquitinated by the anaphase-promoting complex APC and degraded during mitosis McGarry and Kirschner, ; Wohlschlegel et al.
In Xenopus and Drosophila early embryonic cell cycles, however, Geminin is present throughout the cell cycle Quinn et al. In this situation, Geminin is prevented from interacting with Cdt1 during interphase Tada et al. However, in contrast to other APC substrates that are polyubiquitinated, Geminin inactivation does not require degradation by the proteasome Li and Blow, Since the ubiquitination is transitory, a second modification of Geminin may occur after ubiquitination that inactivates Geminin.
Therefore, in addition to negatively regulating licensing by inhibiting pre-RC formation, CDK positively regulates licensing by inactivating Geminin upon exit from metaphase.
Geminin homologues have not been identified in yeast. Since Geminin is also involved in development Kroll et al. The importance of Geminin in regulating licensing and maintaining genomic stability was demonstrated by knockdown experiments in Drosophila and mammalian cells where reduction of Geminin results in rereplication and checkpoint activation that prevents entry into mitosis Mihaylov et al. Therefore, Geminin's inhibitory function may be specific for S phase when CDK levels are not high enough to suppress relicensing, whereas in mitosis CDK is primarily responsible.
It has been suggested that, in addition to its role as a negative regulator of licensing in S phase, Geminin plays a second role as a positive regulator of licensing during M phase by binding to and stabilizing Cdt1 Ballabeni et al. While Geminin knockdown in S phase induces rereplication, knockdown of Geminin in M phase resulted in impaired pre-RC formation during the ensuing cell cycle.
It will be interesting to determine if post-translational modification of Geminin is responsible for the different activities of Geminin. It is important to mention that depletion of Geminin in cycling Xenopus extracts fails to stimulate rereplication McGarry and Kirschner, However, Geminin depletion does result in a G2 arrest during embryonic cell cycles dependent on Chk1 McGarry, similar to that seen in Drosophila and mammals.
Therefore, it is possible that a very low level of reinitiation occurs that is sufficient for checkpoint activation, but not significant enough to be detected. Another possibility is the presence of Geminin-independent mechanisms that prevent rereplication, which are unique to Xenopus embryonic cell cycles. Defects in replication initiation lead to halting of cell cycle progression through what are known as checkpoint pathways Nyberg et al.
Checkpoints ensure that cell cycle events have occurred correctly and completely before proceeding to the next stage of the cell cycle. Furthermore, checkpoints facilitate repair and induce programmed cell death in the face of irreparable damage. As a result, cells defective for checkpoint proteins are highly sensitive to damaging agents and cellular stress. A subset of checkpoint proteins are essential for viability in the absence of exogenous damage, indicating that checkpoint proteins are also required to deal with endogenous stresses that occur during normal cell proliferation.
In this section, we will cover checkpoints that are activated by disruption of the replication process; in particular, checkpoints activated by stalled forks and inappropriate licensing. Checkpoints activated by irradiation and double-stranded breaks will not be discussed; however, the different checkpoints often activate parallel and overlapping pathways that feed into common downstream effectors. We also defer discussion of specialized DNA polymerases involved in tranlesion synthesis to other reviews Friedberg et al.
Elongating replication forks stall when nucleotide pools are depleted or when they encounter lesions in DNA such as those caused by the alkylating agent MMS. Replication forks appear to be the sensor for detecting damage caused by alkylating agents as checkpoints are activated only during S phase regardless of when MMS is added during the cell cycle Tercero and Diffley, ; Tercero et al.
Experiments in Xenopus also show that replication is required for checkpoint activation during S phase Lupardus et al. Therefore, damage may go undetected until a replication fork collides with the lesion. Stalled replication forks signal a cascade of events that inhibit origin firing, stabilize stalled forks and prevent entry into mitosis Nyberg et al.
The most important function for maintaining cell viability is the stabilization of stalled replication forks Desany et al. Maintaining the integrity of forks is imperative for survival because restarting stalled forks is the primary mechanism for recovering from damage Lopes et al.
Therefore, stalled replication forks are both the primary sensor and target of checkpoints. In checkpoint-deficient yeast strains, stalled replication forks undergo irreversible transformations to abnormal structures that are unable to restart DNA synthesis Lopes et al. Some of these structures resemble substrates for recombination intermediates, suggesting that in the absence of fork stabilizing mechanisms inappropriate recombination occurs, leading to chromosome instability Sogo et al.
Similar findings are observed in mammalian cells where checkpoint inhibition releases replication factors from chromatin, presumably due to fork collapse, and prevents stalled forks from resuming synthesis Dimitrova and Gilbert, Recent work, mainly carried out in yeast, has begun to clarify the mechanism by which stalled forks activate these pathways Figure 3.
Stalled forks generate stretches of single-stranded DNA Sogo et al. ATR is a central player in multiple checkpoint pathways that are activated by damage in addition to those caused by stalled forks, suggesting that different forms of insults are processed to a common intermediate such as single-stranded DNA. Interestingly, two of these proteins, Mrc1 and Tof1, travel with the normal elongating replication fork and are loaded after Cdc45 during origin firing Katou et al.
The metazoan homologue of Mrc1, Claspin, is similarly loaded at origins during replication initiation Lee J et al. No homologue of Tof1 in higher eukaryotes has been described thus far.
Other components of the replication fork also have roles in mediating the checkpoint signal in addition to their function in initiation and elongation such as Dpb11 Araki et al. It is unclear how these checkpoint pathways stabilize stalled replication forks. Checkpoints may prevent the premature disassembly of the replication machinery as loss of Mec1 results in dissociation of Cdc45 Katou et al. It has also been postulated that Rad53 may prevent decoupling of the replicative polymerases that occur when forks stall Sogo et al.
Checkpoint activated in response to stalled replication forks. Nucleotide depletion or DNA damage causes replication forks to stall, resulting in accumulation of single stranded DNA. RPA-coated single-stranded DNA recruits Mec1 through Ddc2 to phosphorylate downstream components of the checkpoint pathway that function to stabilize replication forks and halt cell cycle progression.
Tof1 and Mrc1 are components of the normal replication fork that are necessary for propagating the checkpoint signal. Depicted in the figure are the names of proteins from S. In addition to propagating the checkpoint signal, Mrc1 and Tof1 play a role in pausing replication forks in the presence of hydroxyurea Katou et al. Failure to pause in Mrc1 and Tof1 mutants results in the migration of the replication machninery along the chromosome without DNA synthesis.
However, it is not known whether this also occurs in response to alkylating agents which present a physical barrier to fork migration.
The finding that the replication fork has evolved to incorporate checkpoint proteins suggests that fork pausing and restarting may occur during a normal S phase as the replication machinery encounters higher-order chromatin structures, or in response to endogenous sources of damage such as free radicals generated during normal metabolic processes. Rereplication can be produced in cancer cells by overexpression of Cdt1 and Cdc6 Vaziri et al. Rereplication caused by depletion of Geminin in mammalian cells also activates a checkpoint that prevents cell cycle progression.
However, the exact nature of the molecular signal that activates the checkpoint remains to be determined, nor is it clear as to why p53 or Cdc25C are differentially used as effectors in response to rereplication induced by Cdt1 overexpression versus geminin depletion.
The purpose of these pathways is to maintain genomic integrity as ablation of these checkpoints results in a catastrophic mitosis with chromosomal breakage Melixetian et al.
Therefore, loss of Geminin may predispose cells to chromosomal instability and cancer. In addition to relicensing, the effects of insufficient licensing were examined in various mammalian cell lines by overexpressing a nondegradable form of Geminin Shreeram and Blow, Interestingly, cancer cell lines and primary cell lines responded differently to the inhibition of licensing.
Cancer cell lines entered S phase and underwent apoptosis with activation of S phase checkpoints. However, a primary cell line arrested in G1 with low levels of CDK activity. It will be interesting to determine the signals that trigger checkpoint activation in response to inappropriate or inadequate licensing, and whether it contributes to development of cancer.
A growing body of evidence has associated replication factors with other cellular processes, probably as a means of communicating replication status to the cell cycle. A good example is the replication proteins that are required for both initiation and checkpoint activation leading to cell cycle arrest. Although ORC is required for localizing pre-RC components to origins in all eukaryotes, recent studies have identified additional functions for several of the ORC subunits.
Orc6 has been shown to be involved in chromosome segregation and cytokinesis Prasanth et al. These functions occur outside the context of the six-subunit ORC complex, suggesting that the different subunits may have modular functions which can be incorporated into other complexes.
Consistent with this, subsets of the six human ORC subunits have been reported to be present in nonproliferating tissues like the heart or the brain Thome et al. ORC is also involved in heterochromatin formation and transcriptional silencing separable from its role in replication Foss et al.
Geminin has been shown to be important for regulating the function of replication proteins and transcription factors during development Del Bene et al. No doubt, more of these relationships will be uncovered in the future. Since the discovery of the structure of DNA 50 years ago which produced a mechanism for copying the genome, and the purification of DNA polymerase which provided the molecular means to accomplish the task, scientists have been trying to understand how the two come together to faithfully duplicate the genome with every cell division.
We now know that a highly regulated pathway of protein interactions need to occur before polymerase is positioned to begin DNA synthesis. Although we continue to identify many of the players involved, we know very little about the biochemical activities of these proteins at an origin, and how they contribute at the molecular level to the replication process.
A major challenge in the future will be to decipher these mechanisms using a combination of structural studies and biochemistry. Another fundamental question that remains unanswered is what are the genetic and epigenetic elements that define an origin?
Answering this question will be aided by the completed sequence of several eukaryotic genomes, as we are now capable of performing high resolution mapping across chromosomes to identify and analyse origins on a larger scale. These kinds of studies may also uncover clues to understanding the problem of origin timing and spacing, and provide insight into how origin selection and replication fork progression are influenced by factors like gene density, epigenetic changes and the genetic background of the cell.
The recent breakthrough of RNAi technology has provided scientists with the ability to perform genetics in mammalian cells. Genetic studies in yeast have been instrumental for understanding the replication process, and, although many of the proteins involved are conserved, yeast and humans are separated by a billion years in evolutionary distance. Genetic screens will be a powerful resource for characterizing processes unique to mammalian replication. The replication initiation factors and regulators described here are expected to become important for developing new therapies for cancers.
The interaction of the disorders of replication initiation with checkpoint pathways clearly needs further exploration. Given that mutations in checkpoint pathways and overexpression of replication initiator proteins are seen in human malignancies, it is likely that an understanding of these interactions will reveal how they contribute to genetic instability in cancer cells.
Conversely, efforts are under way to take advantage of the checkpoint pathway anomalies in cancers for therapeutic benefit. Levels and activities of replication factors and their regulators might then have prognostic significance for such therapeutic interventions.
Finally, some of the replication initiator proteins have been implicated in the replication of viral episomes in cancer cells Dhar et al. Further work is needed to understand this dependence and to exploit it to develop therapies for cancers that are caused by episomally maintained viruses. Genes Cells , 2 , — Nature , , — Cell Biol. Cell , , — USA , 96 , — Cell , 91 , 59— S Phase stands for Synthesis Phase. It is then followed by division of the cell.
If the s- phase fails to occur, a checkpoint before the division phase prevents the further proceedings of the cell cycle. Hence without the happening of S-phase, cell division will never complete. Telophase does not belong. G1 phase, G2 phase, and S phase are all part of Interphase.
After G1 phase S phase occurs. S phase. The Phase Change is complete, e. The S phase is the synthesis phase and this is when the genetic material, DNA, is replicated. S phase of a cell cycle is The replication phase of a cell S stands for synthesis. This is the phase in which the DNA is replicated in advance of the phases of mitosis. S phase A complete cardiac cycle. The S synthesis phase is important because this is the phase in which DNA is replicated.
This is essential so that cell replication can occur. DNA replication happens during s-phase. Chromatin DNA in loose form is replicated in the S phase of interphase.
Interphase follows prophase and is comprised of a G1 phase, S phase, and G2 phase. Log in. Study now. See Answer. Best Answer. Study guides. Genetics 22 cards. What is a chromosome. What is an allele. Currently, many researchers are trying to better understand the mechanisms of crosstalk between ATR and the replication machinery Forsburg ; Bailis et al.
Nature Reviews Molecular Cell Biology 9 , doi So why would normal cells need ATR? There are other circumstances that cause replication to go awry. One is that the DNA template somehow becomes defective during replication, and causes the polymerase to pause Figures 3 and 4a.
For example, a DNA base can be chemically modified or spontaneously altered. Scientists use the term "stalled forks" for areas of replication forks where DNA polymerization is halted. Little is known about the phosphorylation targets that lie further downstream of Chk1, but when scientists observe Chk1 phosphorylation in cells, they conclude that cells are actively trying to protect replication forks with DNA lesions.
What happens when ATR function goes awry? A DSB is a catastrophic event because it ruins the replication fork. Under these circumstances, cells activate the ATM kinase Figure 4, on the right. It does so by phosphorylating checkpoint kinase 2 Chk2 , a protein that triggers a cascade of phosphorylation events that ultimately result in the repair of the DSB. Interestingly, when Chk2 triggers events that ultimately repair a DSB, another event also takes place.
This event is the phosphorylation of the well-known p53 Caspari This observation is a clue that repairing DSBs may have something to do with preventing the formation of tumors. Together with a variety of other molecules, ATR and ATM kinases are key factors for the surveillance of DNA replication, and prevent chromosome breakage in dividing cells.
However, during repair processes, chromosome fragments can be improperly joined together. Indeed, some scientists consider that such mistakes enable some degree of genetic evolution by creating new and different genetic sequences.
Nevertheless, if even a single cell in our body makes a mistake and fuses DNA fragments to each other that are not supposed to be joined, the rearrangement can be sufficient to deregulate normal cell division. If multiple changes of this type accumulate, then this single cell can eventually turn into a tumor.
In these affected individuals, the cellular surveillance system described above is defective and no longer provides full protection from random events that affect DNA replication. For example, the name of the ATM protein derives from the affliction that results from a mutated ATM protein: ataxia telangiectasia.
In this disease , patients suffer from motor and neurological problems, and they also have what is known as a genome instability syndrome that genetically predisposes them to developing cancer Shiloh With these observations, it may be possible to create new ideas for novel diagnostics and therapies for cancer that specifically track these potent molecules.
The process of DNA replication is highly conserved throughout evolution. Investigating the replication machinery in simple organisms has helped tremendously to understand how the process works in human cells. Major replication features in simpler organisms extend uniformly to eukaryotic organisms, and replication follows fundamental rules.
During replication, complex interactions between signaling and repair proteins act to keep the process from going awry, despite random events that can cause interruption and failures. Discovering the exact repair mechanisms that help keep DNA intact during replication may help us understand the mechanisms of tumor growth, as well as develop strategies to detect or treat cancer.
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The Journal of Biological Chemistry — Forsburg, S. The MCM helicase: linking checkpoints to the replication fork. Biochemical Society Transactions 36 — Gambus, A. Hubscher, U. Eukaryotic DNA polymerases. Annual Review of Biochemistry 71 — doi Katou, Y. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature — doi Kouprina, N. Molecular and Cellular Biology 12 — Langston, L. DNA replication: keep moving and don't mind the gap.
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