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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Wold M. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Alani E. Siegal G. Goulian M. Discontinuous DNA synthesis by purified mammalian proteins. Waga S. Budd M. Temperature-sensitive mutations in the yeast DNA polymerase I gene. Sitney K. Boulet A. EMBO J. Morrison A. A third essential DNA polymerase in S. Fisher P. Basic catalytic properties processivity, and gap utilization of the homogeneous enzyme from human KB cells.
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Nishitani H. Control of DNA replication licensing in a cell cycle. Genes Cells. Lei M. Cell Sci. Eukaryotic DNA replication origins: Many choices for appropriate answers. Cell Biol. Coleman T.
The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. Tanaka T. Ogawa Y. Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins. Maiorano D. XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. The Cdt1 protein is required to license DNA for replication in fission yeast. Remus D. Evrin C. Dahmann C. S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state.
Mimura S. Zou L. Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin. Nougarede R. Hierarchy of S-phase-promoting factors: Yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation. Sheu Y. The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase.
Masai H. Muramatsu S. Genes Dev. Handa T. DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast. Saxena S. Geminin-Cdt1 balance is critical for genetic stability. Mendez J. Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication.
Diffley J. Two steps in the assembly of complexes at yeast replication origins in vivo. Liang C. Lee K. Phosphorylation of ORC2 protein dissociates origin recognition complex from chromatin and replication origins. Saha P. Wohlschlegel J. Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Hook S. Mechanisms to control rereplication and implications for cancer.
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SUMOylation regulates Radmediated template switch. Ulrich H. Cell Cycle. Leach C. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. There are additional links in Blackboard. Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division.
The process of DNA replication can be summarized as follows:. Table 1: The enzymes involved in prokaryotic DNA replication and the functions of each. DNA replication has been extremely well-studied in prokaryotes, primarily because of the small size of the genome and large number of variants available. Escherichia coli has 4. This means that approximately nucleotides are added per second. The process is much more rapid than in eukaryotes. OpenStax , Concepts of Biology. OpenStax CNX.
Skip to content The prokaryotic chromosome is a circular molecule with a less extensive coiling structure than eukaryotic chromosomes. The process is quite rapid and occurs without many mistakes. DNA replication employs a large number of proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one by one to the growing DNA chain that are complementary to the template strand.
The addition of nucleotides requires energy; this energy is obtained from the nucleotides that have three phosphates attached to them, similar to ATP which has three phosphate groups attached. When the bond between the phosphates is broken, the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain.
How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs.
ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi- directionally as replication proceeds. Single-strand binding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix.
Then how does it add the first nucleotide? Because this sequence primes the DNA synthesis, it is appropriately called the primer. DNA polymerase can now extend this RNA primer, adding nucleotides one by one that are complementary to the template strand [link]. Next Video 6. This complex helps to initially separate the DNA. Then the enzyme DNA helicase binds to it and continues to unwind the DNA by breaking the hydrogen bonds between the complementary strands. The newly opened areas are stabilized by single-stranded DNA binding proteins.
Each one can now serve as a template for the synthesis of a new strand of DNA. The unwinding and synthesis proceeds in both directions from the origin, creating two replication forks. In front of the forks, topoisomerase enzymes bind to the DNA and reduce torsional strain as the molecule unwinds. The primer provides a place for the enzyme DNA polymerase to add nucleotides complementary to the DNA sequence, creating a new DNA strand in a process called elongation.
DNA polymerase synthesizes DNA in the five prime to three prime direction of the molecule, so the synthesis of this strand, the leading strand, proceeds continuously. The other strand, the lagging strand, has the opposite orientation. Consequently DNA is synthesized in short pieces called Okazaki fragments, elongated from additional RNA primers backwards from the overall direction of movement of the replication fork.
This is considered a semiconservative process, because each molecule contains one old strand and one new strand. DNA replication has three main steps: initiation, elongation, and termination.
Replication then proceeds around the entire circle of the chromosome in each direction from two replication forks, resulting in two DNA molecules. Replication is coordinated and carried out by a host of specialized proteins. Topoisomerase breaks one side of the double-stranded DNA phosphate-sugar backbone, allowing the DNA helix to unwind more rapidly, while helicase breaks the bonds between base pairs at the fork, separating the DNA into two template strands.
Proteins that bind single-stranded DNA molecules stabilize the strands as the replication fork travels along the chromosome. Much of the research to understand prokaryotic DNA replication has been performed in the bacterium Escherichia coli , a commonly-used model organism.
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