If the problem continues, please let us know and we'll try to help. An unexpected error occurred. Previous Video 6. These separate, single-stranded DNA molecules are prone to form double-stranded hairpin loops or to rewind with the other strand. Now the exposed single strands of DNA can act as templates for the synthesis of the complementary daughter strands.
The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork. Organisms with small circular DNA, such as E. In organisms with large genomes, the replication of DNA is not done from a single point of origin but in many distinct, localized replication forks.
The unhindered progression of the replication fork is necessary for complete DNA replication and genome stability; however, the replication fork is often stalled by internal or external factors that can slow or stop its progression, resulting in replication stress.
Replication stress causes genomic instability, which is a hallmark of diseases like cancer. Genomic instability is characterized by genomic alterations and increased frequency of harmful mutations. The movement of the replication fork can stop due to several reasons.
For example, the drug hydroxyurea depletes the pool of nucleotides available for incorporation into the daughter strand, stalling the replication fork. Other problems that may hinder the progression of the replication fork include DNA lesions, a collision between a replication fork and a transcription complex, and defects in the enzymes involved in DNA replication. The cell has a variety of repair mechanisms to reinitiate the stalled replication fork.
S-phase checkpoints do not allow cells to begin mitosis until DNA repair is complete. C At the mating-type mat1 locus in fission yeast, Rtf1 bound to the RTS1 site prevents replication fork progression at the 5' side of mat1. Only the fork in the 3' direction can progress through the region. This fork pauses at MPS1 and generates an imprint, probably a DNA strand discontinuity, which is required for a specific recombination event that allows for mating-type switching.
In prokaryotes, such as the Escherichia coli bacterium, bidirectional replication initiates at a single replication origin on the circular chromosome and terminates at a site approximately opposed from the origin [ ]. This replication terminator region contains DNA sequences known as Ter sites, polar replication terminators that are bound by the Tus protein. The Ter -Tus complex counteracts helicase activity, resulting in replication termination [ ]. In this way, prokaryotic replication forks pause and terminate in a predictable manner during each round of DNA replication.
In eukaryotes, the situation differs. Replication termination typically occurs by the collision of two replication forks anywhere between two active replication origins. The location of the collision can vary based on the replication rate of each of the forks and the timing of origin firing.
Often, if a replication fork is stalled or collapsed at a specific site, replication of the site can be rescued when a replisome traveling in the opposite direction completes copying the region. To efficiently terminate or pause replication forks, some fork barriers are bound by RFB proteins in a manner analogous to E. In these circumstances, the replisome and the RFB proteins must specifically interact to stop replication fork progression. Budding yeast chromosome XII contains an array of approximately rDNA repeats, which are highly transcribed in order to produce the ribosomes necessary for translation.
Therefore, transcription forks are exceedingly common in rDNA regions, which can lead to head-on collisions between replication and transcription forks, possibly leading to collapse of one or both forks. To prevent these collisions, replisome progression opposing the direction of ribosome transcription is inhibited at rDNA repeats, thus allowing for replication and transcription to proceed in the same direction through the rDNA loci [ ].
How is this polar replication block achieved? Fob1 operates by looping or wrapping the DNA around itself to dramatically alter the local chromatin state and block replication from the 3' direction [ ]. This Fob1-dependent chromatin structural change not only causes polar replication fork pausing but also leads to increased recombination at the RFB region [ ]. It is proposed that Rrm3 displaces Fob1 to facilitate fork progression [ ].
In addition, Rrm3 is thought to be required for replication restart in other genomic loci requiring displacement of nonhistone proteins from DNA [ , , , , ]. Interestingly, Rrm3 translocates with the replication fork and physically interacts with PCNA [ , ].
Therefore, similar protein-protein interactions between RFB proteins and Rrm3 may allow for polar passage of replication forks through multiple fork block sites throughout the genome.
Although Fob1 is a budding yeast-specific protein, similar 3' specific replication termination occurs in the rDNA array in fission yeast and mammalian cells. The DNA binding protein Sap1, which also recognizes the mating-type locus, causes polar fork arrest at Ter1 , while fork arrest at Ter2 and Ter3 is regulated by another RFB protein, Reb1, which is homologous to mammalian transcription terminator factor 1 TTF-1 [ , , , ].
These proteins act to specifically inhibit polar replication progression, coordinating DNA replication with transcription to minimize fork collisions and genome instability.
In addition to site-specific fork pausing required to prevent collision between replication and transcription machinery, fork pausing also allows for programmed cellular events that are coordinated with replication of specific genomic loci. For example, the fission yeast genome contains two genetically programmed fork pausing sites near the mating-type mat1 locus: the mat1 pausing sites 1 MPS1 and replication termination site 1 RTS1.
A strong polar fork arrest at RTS1 blocks one replication fork moving into the mat1 locus, allowing only the opposing fork to migrate into the mat1 locus. This fork pauses at MPS1 , generating an imprint that initiates a replication-coupled recombination event, leading to mating-type switching Figure 6 C [ , , , , , ]. The RTS1 -dependent fork arrest is DNA sequence-specific, because inserting the RTS1 sequence into other genomic loci in Schizosaccharomyces pombe also results in polar fork arrest and increased recombination at those sites [ , ].
Rtf1 mutations disrupt polar fork arrest, indicating that Rtf1 is required for unidirectional replication termination at the RTS1 loci [ , ].
Interestingly, efficient pausing at RTS1 also requires the presence of specific replisome proteins. The requirement for both replication termination factors on the DNA and replisome components suggests that these proteins may interact to regulate replication pausing or termination at the mat1 locus. Together, these RFB sites provide insights into the role of chromatin-bound proteins in regulating replisome progression at specific loci.
Examining DNA replication on a global level, it appears that some loci are more sensitive to DNA replication stress than others. Analysis of chromosomal DNA using two-dimensional agarose gel electrophoresis, showed that checkpoint mutants accumulate replication forks at specific genomic regions [ ].
This led to the suggestion that certain genomic regions take longer to replicate because they are more difficult to replicate when compared with the rest of the genome. The replication fork-enriched loci were coined replication slow zones RSZs.
This is consistent with the observation that functional checkpoint proteins are generally required to replicate DNA during stress [ ]. Early hypotheses proposed that RSZs were advantageous for general replication, allowing for the replisome to coordinate with other processes e.
Recent work has suggested that mec1 mutant cells fail to replicate through RSZs due to a partial inability to upregulate dNTP levels relieving inhibition of ribonucleotide reductase II by Sml1. Therefore, the primary function of Mec1 may be to act as a sensor at the replication fork, upregulating nucleotide synthesis when there is a demand for increased dNTP levels during DNA replication. Considering that the checkpoint is required even in the absence of exogenous replication stressors, something inherent to RSZs causes replication stress.
As described above, DNA replication through these challenging regions requires checkpoint proteins in budding yeast. This is consistent with replication in the presence of exogenous genotoxic agents such as methyl methanesulphonate MMS: an alkylating agent, which arrests forks or hydroxyurea HU: a ribonucleotide reductase II inhibitor, which depletes cellular dNTP pools and arrests forks.
In the absence of Rad53 checkpoint kinase, budding yeast cells accumulate unusual replication fork structures in response to MMS or HU; thus cells are unable to complete replication [ , ].
In contrast, wild-type cells are eventually able to complete replication in the presence of genotoxic agents, albeit slowly and with continued activation of repair and checkpoint pathways. In MMS-treated cells, DNA replication stress is induced globally, and replication is generally slowed by the checkpoints. In the RSZ model, the stress is induced by replication of the specific genomic region. The replisome encounters difficulties at these regions, resulting in fork arrest and generation of DNA structures that are recognized by checkpoint proteins.
Whether the source of replication stress is exogenous or endogenous, checkpoint proteins recognize stalled forks, upregulate nucleotide synthesis, activate repair pathways, and arrest the cell cycle. By associating with the replisome, checkpoint kinases are well positioned to send signals to cope with fork arrest in response to a multitude of replication stressors, including endogenous obstacles on DNA sequences and exogenous genotoxic agents Figure 7.
Replication stress is induced by multiple factors in vivo. Some stresses are relatively rare events such as DNA damage, while others must be encountered at the same genomic sites every S phase, such as difficult-to-replicate regions. Recurrent DNA breaks that can be visualized on metaphase chromosomes as breaks and gaps are known as fragile sites. Fragile sites lead to recurrent breaks in families and were initially characterized as DNA breakage sites inherited in a manner predicted by Mendelian genetics; these breaks could be visualized on metaphase chromosomes and mapped to specific chromosome arm regions [ ].
Further investigations have connected the occurrence of fragile sites with human diseases such as fragile X syndrome, the most common heritable form of mental retardation [ ]. In cultured cells from normal individuals, these sites are relatively stable, and chromosome breakage rarely occurs. However, breakage at these sites can be induced by depleting folate or by adding excess thymidine in cultured human cell lines [ , , ]. Because nucleotide repeats are considered to be poor templates for DNA polymerases and can induce replication fork stalling, breakage at rare fragile sites are likely to occur during DNA replication [ , , ].
Other studies have reported additional fragile sites using bulky nucleotide analogues, such as 5-aza-cytidine or bromodeoxyuridine [ ]. These sites are less well characterized than aphidicolin-dependent fragile sites, but both types of fragile sites are found in AT-rich genomic loci. Thus, chromosome breakage at specific loci is often due to replication perturbations, which are generally provoked by inherent features associated with the specific chromosomal loci.
Fragile sites appear to share common features that may help to understand the mechanisms of chromosome breakage. Breakage rates at aphidicolin fragile sites are dependent on drug concentrations, and these sites tend to form secondary structures and replicate late in S phase [ , , ]. Because common fragile sites occur in AT-rich repeat regions, it is possible that the properties of AT-rich DNA hinder proper replication processes.
For example, AT-repeat rich microsatellites contain sequences that are structurally flexible with regard to base pairing and can form hairpin secondary structures [ ]. Interestingly, all fragile sites including common and rare share a late-replicating phenotype [ ]. It is possible that late S-phase replication of fragile sites allows for proper DNA replication of difficult templates. However, conversely, it is also possible that cells delay replication of fragile sites owing to the inherent difficulty of replicating the region.
As a consequence, these fragile sites may break due to diminished dNTP pools at the end of S phase, thus requiring checkpoint proteins to complete the region. Other factors may play a role in the breakage of fragile sites. However, DNA breakage at the site was not entirely abrogated, implying that chromatin context, not simply DNA sequence, also contributes to breakage propensity.
Additionally, ATR has been shown to be required for replication through these regions, suggesting intimate regulation of DNA replication through these regions by the cell cycle checkpoint [ ]. Global mapping and characterizations of these sites will reveal how various features of fragile sites contribute to their challenging replication phenotype.
The study of eukaryotic DNA replication has continued to expand over the past several decades and will likely continue to do so, filling the knowledge gaps in the regulation of replication processes. Many of the recent advances in the field have focused on how specific DNA sequences, structures, and regions are specifically regulated.
Further studies will elucidate how characterized and yet-unidentified replisome proteins contribute to replication processes in a site-specific manner. In our current understanding, the replisome and replisome-associated factors are able to respond to a variety of hindrances to successfully complete DNA replication Figure 7.
The above review of challenging loci for replication is by no means exhaustive, and new techniques will likely identify even more genome regions that require specialized mechanisms for successful and efficient replication. Genome-wide approaches will allow us to understand the genomic regions and features affected by perturbation of specific replisome components.
Current developments in single-molecule studies will open opportunities to test mechanistic models of replication in a loci-specific manner. The next few years should see great advances in our understanding of replication regulation at the global and locus level. Future studies will uncover the mechanisms by which checkpoint proteins and replisome-interacting factors cooperate together to ensure replisome progression of difficult-to-replicate genomic regions. We apologize to the authors of many studies that we could not include in this review owing to space limitations.
National Center for Biotechnology Information , U. Journal List Genes Basel v. Genes Basel. Published online Jan Adam R. Author information Article notes Copyright and License information Disclaimer. This article has been cited by other articles in PMC. Abstract Eukaryotic cells must accurately and efficiently duplicate their genomes during each round of the cell cycle. Keywords: DNA replication, replisome, replication fork, genome stability, checkpoint, fork barriers, difficult-to-replicate sites.
Open in a separate window. Figure 1. Figure 2. DNA Polymerases and Helicases The two basic processes of DNA replication are unwinding of the template strand and polymerization of the daughter strands. Replication Initiation at Origins To completely duplicate the genome in a reasonable time during the cell cycle, eukaryotic cells initiate DNA replication at multiple sites during DNA replication, whereas prokaryotic replication initiates at a single locus.
Figure 3. Figure 4. Replication Checkpoint Proteins In order to preserve genetic information every time the cell divides, DNA replication must be completed with high fidelity. Replication through Nucleosomes Eukaryotic genomes are substantially more complicated than the smaller and unadorned prokaryotic genomes. Figure 5. Figure 6. Replication Fork Barriers In prokaryotes, such as the Escherichia coli bacterium, bidirectional replication initiates at a single replication origin on the circular chromosome and terminates at a site approximately opposed from the origin [ ].
Replication Termination at the Fission Yeast Mating-Type Locus In addition to site-specific fork pausing required to prevent collision between replication and transcription machinery, fork pausing also allows for programmed cellular events that are coordinated with replication of specific genomic loci.
Figure 7. Fragile Sites Recurrent DNA breaks that can be visualized on metaphase chromosomes as breaks and gaps are known as fragile sites. Conclusions and Closing Remarks The study of eukaryotic DNA replication has continued to expand over the past several decades and will likely continue to do so, filling the knowledge gaps in the regulation of replication processes.
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Replication checkpoint protein Mrc1 is regulated by Rad3 and Tel1 in fission yeast. Eukaryotes and Cell Cycle. Cell Differentiation and Tissue. Cell Division and Cancer. Cytokinesis Mechanisms in Yeast. Recovering a Stalled Replication Fork. Aging and Cell Division. How helpful was this page? What's the main reason for your rating? Which of these best describes your occupation? What is the first part of your school's postcode?
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