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.
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EMBO Rep. Each genome contains all of the information needed to build that organism and allow it to grow and develop. DNA or deoxyribonucleic acid is a long molecule that contains our unique genetic code. Like a recipe book it holds the instructions for making all the proteins in our bodies. Cells are the basic building blocks of living things. The human body is composed of trillions of cells, all with their own specialised function. If you have any other comments or suggestions, please let us know at comment yourgenome.
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