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. Can you spare minutes to tell us what you think of this website? Browse Visually. Other Topic Rooms Genetics. Student Voices. Creature Cast.
Simply Science. Green Screen. Green Science. Bio 2. The Success Code. Why Science Matters. The Beyond. Plant ChemCast. Postcards from the Universe. Brain Metrics. Mind Read. Eyes on Environment. Accumulating Glitches. Saltwater Science. Microbe Matters. You have authorized LearnCasting of your reading list in Scitable.
Do you want to LearnCast this session? This article has been posted to your Facebook page via Scitable LearnCast. These findings shed new light on how the DNA replication program and the proliferation rate are being modulated during cell fate commitment, when cell type specialization is determined. New discoveries in this topic are undoubtedly essential to expand our understanding about the genesis of diseases and their progression. In this review, we will briefly describe recent achievements aiming to understand how the replication factors involved in the licensing, activation, and elongation steps of DNA synthesis adapt dynamically with cell fate determination to maintain genome integrity and homeostasis.
In vitro reconstitution assays in yeast have provided tremendous detailed information on how origins are licensed by dissecting the sequential biochemical steps involved 3 , 7 — 9 , 14 , Detailed reviews covering the main features that characterize metazoan origins have been recently published 3 , 5 , Briefly, in eukaryotes, replication origins are licensed through a chronological order that requires the binding of Origin Recognition Complex ORC , Cdc6, and Cdt1 proteins onto chromatin 17 , Two hexamers of the MCM helicase complex then are loaded in an inactive state prior to S phase Figure 1.
The choice of the origins to be activated around 30, in mammalian cells is variable from cell to cell and ensures flexibility in origin usage during the DNA replication program to adapt with cell fate commitment, cell environment, and replicative stress 22 , Origin mapping strategies coupled with high-throughput sequencing revealed that the chromatin context influences origin selection through genetic and epigenetic features located in close proximity to the origins.
Replication origins are scattered along the genome to ensure that each chromosome is entirely replicated in S phase. Pre-replication complex Pre-RC assembly and origin activation are tightly regulated in a sequential manner to ensure that replication occurs only once per cell cycle. Once a double MCM hexamer is stably recruited onto chromatin, origins are licensed. Many replication origins are licensed but few are activated, allowing a backup of origins to be used when DNA replication is perturbed.
The entire replication machinery made of accessory factors required for replication fork stability and the synthesis of DNA is called the replisome. Notably, this population of shared origins tends to initiate early during S phase, whereas cell type—specific origins initiate during late S-phase and are linked with compacted chromatin marks These observations underline the relationship between chromatin modifications with the cellular context in origin selection.
Two independent studies describing reconstitution of DNA replication initiation events in vitro from chromatinized templates in yeast provided biochemical evidence that the regulatory functions of chromatin structure influence origin selection. These studies confirmed that nucleosome-free regions contribute to defining ORC binding and thus origin function 30 , 31 , as previously suggested from genome-wide studies mapping ORC binding sites and nucleosome occupancy in various organisms 32 — Moreover, in mouse embryonic stem ES cells, depletion of the histone H1 perturbs the landscape of replication origin activation Because chromatin environment changes during cell fate commitment and in different cell types, it is generally assumed that the DNA replication program is coordinated with transcription to avoid transcription—replication conflicts and thus preserve genomic integrity.
This seems particularly crucial during the onset of developmental programs and cell lineage specification. In Caenorhabditis elegans , origins from rapidly replicating pluripotent embryos coincide with open chromatin regions of highly transcribed genes, whereas establishment of the new transcriptional program, occurring at later embryonic stages when cell differentiation begins, correlates with the reorganization of replication initiation sites 29 , Permissive chromatin features for rapid activation of replication origins could influence the cell cycle length.
ES cells have a shorter G 1 compared with their differentiated counterparts, which impacts on the licensing step. ES cells recruit much more MCM to the chromatin than tissue-specific stem cells or progenitor cells This excess of origins that remain dormant during S phase appears to be important to maintain pluripotency 40 , 41 and could also protect the genome against DNA replication stress occurring in ES cells 39 , Besides changes in the kinetics by which MCM is loaded onto chromatin, licensing control adapts to G 1 length, suggesting that pre-RC binding is developmentally regulated.
Indeed, quantitative single-cell analysis performed in human cells by Matson and colleagues demonstrated that origins are licensed faster in pluripotent cells compared with their isogenic differentiated counterparts and that loading of the MCM helicase slows down as G 1 duration is extended The authors revealed that high expression of the licensing factor Cdt1 was important for fast MCM loading rates, thus enabling ES cells to rapidly license origins prior to the G 1 -to-S phase transition.
Similar to the need for additional dormant origins, fast licensing kinetics seems essential to ensure pluripotency in ES cells and induced pluripotent stem cells. This observation was corroborated by Carroll and colleagues in intestinal stem cells from adult tissues The authors demonstrated that licensing is interconnected with the proliferative commitment of stem cells.
Interestingly, this unlicensed state correlates with an elongated cell cycle that could be considered as a backup mechanism to sustain proliferative fate decisions and tissue maintenance. Nonetheless, the link between chromatin structures and adaptation to G 1 length in such a context remains to be determined. Uncoupling of G 1 length with licensing kinetics has been observed during oncogenic transformation. Overexpression of oncogenes, such as cyclin E and c-MYC, shortens G 1 and forces cells to engage S phase earlier with incomplete licensing.
Consequently, the replication program is perturbed, leading to accumulation of replication stress 45 , Origin usage following oncogene activation was recently investigated genome-wide. The authors found that in these conditions new initiation zones, which were normally suppressed by transcription in G 1 , appear in intragenic regions These results also confirm an observation in drosophila showing that active transcription modulates MCM distribution An emerging picture from these studies is that cell cycle length, local chromatin structure, and active transcription can modulate the extent of origin licensing.
Chromatin environment contributes to licensing as described previously through MCM loading but also to origin selection through MCM activation 51 , The dynamics of origin activation during S phase follow a spatio-temporal order known as replication timing Figure 2.
Replication timing in eukaryotes controls activation of replication of large chromosomal domains and is mediated by genetic determinants, local histone modifications, and global chromatin organization Thus, DNA sequence features that mediate licensing in vertebrates can also affect the time when origins will be activated.
Their presence in the genome is associated with origin activity 25 , Nucleosome organization at the proximity of origins was reported to modulate both origin licensing as described above and MCM helicase-dependent activation steps of initiation 55 in yeast and more recently in mammals Different histone modifications and certain histone modifiers have been assigned in origin selection and the dynamics of their activation across S phase 29 , 38 , 52 , 56 — Changes in the DNA replication program are dependent on cell features.
In those rapidly dividing embryos, which are transcriptionally inactive, many replication origins fire to ensure fast S-phase. How does the cell duplicate two strands of identical DNA copies simultaneously? The goal of replication is to produce a second and identical double strand.
This is accomplished by a DNA helicase. Once the DNA template is single-stranded ss , a DNA polymerase reads the template and incorporates the correct nucleoside-triphosphate in the opposite position Figure 1.
Because of the characteristic y-shape of the replicating DNA, it is often referred to as a " replication fork. The DNA code in each of the strands is the same, but inverted, so that the sequence is identical when read in the 5' to 3' direction. This is the direction in which all DNA is polymerized, and also the direction in which a DNA sequence is read when written out, by convention.
Figure 1: The major replication events in a prokaryotic cell A Nucleoside triphosphates serve as a substrate for DNA polymerase, according to the mechanism shown on the top strand.
Each nucleoside triphosphate is made up of three phosphates represented here by yellow spheres , a deoxyribose sugar beige rectangle and one of four bases differently colored cylinders. The three phosphates are joined to each other by high-energy bonds, and the cleavage of these bonds during the polymerization reaction releases the free energy needed to drive the incorporation of each nucleotide into the growing DNA chain.
The reaction shown on the bottom strand, which would cause DNA chain growth in the 3' to 5' chemical direction, does not occur in nature. B DNA polymerases catalyse chain growth only in the 5' to 3' chemical direction, but both new daughter strands grow at the fork, so a dilemma of the s was how the bottom strand in this diagram was synthesized.
The asymmetric nature of the replication fork was recognized by the early s: the leading strand grows continuously, whereas the lagging strand is synthesized by a DNA polymerase through the backstitching mechanism illustrated.
Thus, both strands are produced by DNA synthesis in the 5' to 3' direction. All rights reserved. Figure Detail. The DNA strand that is synthesized in the 5' to 3' direction is called the leading strand.
The opposite strand is the lagging stand, and although it is also synthesized in the 5' to 3' direction, it is assembled differently. As a rule, none of the known DNA polymerases adds a nucleoside triphosphate onto a free 5' end. This brings us to the first rule of DNA replication: DNA synthesis only occurs in one direction, from the 5' to the 3' end. Applying this rule helps us understand why the lagging strand is generated from a series of smaller fragments Figure 1b.
These fragments are known as Okazaki fragments, after Reiji and Tsuneko Okazaki, who first discovered them in Each time the DNA fork opens, the leading strand can be elongated, and a new Okazaki fragment is added to the lagging strand. Amongst the array of proteins at the replication fork, DNA polymerases are central to the process of replication.
These important enzymes can only add new nucleoside triphosphates onto an existing piece of DNA or RNA ; they cannot synthesize DNA de novo from scratch , for a given template. Another class of proteins fills this functional gap.
This particular feature of de novo synthesis is similar to what happens during mRNA transcription. This unique enzyme complex is called DNA primase. The chemical properties of DNA and RNA are quite different, and DNA is the preferred storage material for the genetic information of all cellular organisms, so this reinstallment of a continuous DNA strand is very important. But it is likely that some connector protein coordinates DNA unwinding and DNA synthesis initiation in eukaryotic cells.
Figure 2: Proteins at the Y-shaped DNA replication fork These proteins are illustrated schematically in panel a of the figure below, but in reality, the fork is folded in three dimensions, producing a structure resembling that of the diagram in the inset b.
Focusing on the schematic illustration in a, two DNA polymerase molecules are active at the fork at any one time. One moves continuously to produce the new daughter DNA molecule on the leading strand, whereas the other produces a long series of short Okazaki DNA fragments on the lagging strand. Both polymerases are anchored to their template by polymerase accessory proteins, in the form of a sliding clamp and a clamp loader. The helicase exposes the bases of the DNA helix for the leading-strand polymerase to copy.
For this reason, the lagging strand polymerase requires the action of a DNA primase enzyme before it can start each Okazaki fragment. Finally, the single-stranded regions of DNA at the fork are covered by multiple copies of a single-strand DNA-binding protein, which hold the DNA template strands open with their bases exposed.
In the folded fork structure shown in the inset, the lagging-strand DNA polymerase remains tied to the leading-strand DNA polymerase. This allows the lagging-strand polymerase to remain at the fork after it finishes the synthesis of each Okazaki fragment. As a result, this polymerase can be used over and over again to synthesize the large number of Okazaki fragments that are needed to produce a new DNA chain on the lagging strand. In addition to the above group of core proteins, other proteins not shown are needed for DNA replication.
These include a set of initiator proteins to begin each new replication fork at a replication origin, an RNAseH enzyme to remove the RNA primers from the Okazaki fragments, and a DNA ligase to seal the adjacent Okazaki fragments together to form a continuous DNA strand.
In eukaryotic cells, these polymerases cooperate with a sliding clamp called p roliferating c ell n uclear a ntigen PCNA. There may be additional, yet undiscovered, parallel or identical mechanisms or proteins that coordinate DNA unwinding and DNA elongation.
A simple yet often effective approach is to find proteins that directly bind to both enzymes. However, that requires us to understand the molecular architecture of DNA helicase. In eukaryotes, the DNA helicase is comprised of a structural core and two regulatory subunits.
Mcm encircles dsDNA Remus et al. Those factors are cell division cycle protein 45 Cdc45 and GINS Go, Ichi, Ni, and San; Japanese for "five, one, two, and three," which refers to the annotation of the genes that encode the complex. Scientists have actually identified two candidate connector proteins that directly bind to both helicase and primase: 1 Mcm10 another Mcm protein that, despite its name, has no functional resemblance to any of the Mcm proteins Solomon et al.
In budding yeast , Mcm10 is essential for replication to occur. However, in these same cells DNA replication can function normally without Ctf4, which means that Ctf4 is not absolutely required Kouprina et al. What about higher eukaryotes? Other experiments in human cells have shown that both proteins seem to be necessary, and work together during replication Zhu, et al. Scientists are still actively investigating these complex mechanisms.
Why is coordination between DNA unwinding and synthesis important?
0コメント