- Whenever a cell divides, the DNA polymerases copy the genetic information contained in the cell’s lengthy strands of DNA.
- Each strand of DNA produces a template that DNA polymerases use to synthesise a complementary strand.
- At the replication fork, one strand is synthesised continuously in the 5′ to 3′ direction (leading strand), whereas the second strand is synthesised discontinuously in the 3′ to 5′ direction in tiny fragments known as Okazaki fragments (lagging strand).
- The Japanese molecular researchers Reiji and Tuneko Okazaki are credited for discovering these fragments in the 1960s, along with the assistance of a few of their colleagues.
Okazaki Fragments Definition
Okazaki fragments are the DNA fragments that result from the discontinuous replication of the lagging strand.
- The length range of these fragments in bacterial cells is between 1000 and 2000 nucleotides, whereas in eukaryotic cells it is between 100 and 200 nucleotides.
- The Okazaki fragments on the lagging strand are put together to make a new molecule of DNA that is continuous.
- The Okazaki fragments were identified by pulse-labeling E. coli with 3H-thymidine under conditions that greatly slowed the pace of cell growth and division.
- E.coli were cultivated at 37°C for multiple generations in the presence of 14C-thymidine in order to label their DNA evenly with 14C.
- The cells were then cooled to 20°C and pulse-labelled with 3H-thymidine for 10s to label the nascent DNA under conditions where a slower rate of DNA replication could reveal the presence of temporary intermediates.
- The doubling time of E.coli is around 40 minutes at 37°C and approximately 250 minutes at 20°C.
- In the pulse-chase experiments, a large amount of unlabeled thymidine was added to cells that had been pulse-labeled for 10 s at a temperature of 20°C, and incubation continued for the specified durations.
- The net cellular DNA was then extracted and fractionated using alkaline sucrose gradients to completely denature it.
- In each section of the gradient, the amount of acid-insoluble radioactivity that could be rendered soluble by treatment with deoxyribonuclease was determined.
- In such conditions, the bulk of 3H-DNA initially emerged as fragments ranging from around 50 to 5000 nucleotides in length, which rapidly became extended 3H-DNA fragments, arranged with a portion as temporary intermediates in the replication of DNA.
Okazaki Fragments Overview
- During DNA replication in eukaryotic cells, small single-stranded DNA segments known as Okazaki fragments are synthesised first on the lagging strand.
- These fragments arise from RNA-DNA primers that are 35 nucleotides in length. Once the Okazaki fragments have been synthesised, the primers must be eliminated to allow the fragments to form continuous lagging strands.
- Lack of methods that can evaluate the removal of primer directly in vivo is an impediment to elucidating Okazaki fragment processing.
- At a replication fork within a replication bubble, DNA synthesis is semi-interrupted.
- At the replication fork, both daughter DNA molecules are produced. As the two DNA strands are antiparallel, one new strand must be synthesised in a 5′ to 3′ direction, in the same direction as the fork approaches, while the other strand must be synthesised in an overall 3′ to 5′ direction, relative to the movement of the fork.
- DNA polymerases are the enzymes which catalyse the addition of deoxyribonucleotides to an expanding chain of DNA.
- How is the new strand aligned as 3′ to 5′ in the direction of the replication fork if there is no 3′ to 5′ synthesising activity? The solution is the discontinuous synthesis of this strand, although the other strand is continually synthesised.
- Now that one of the template DNA strands is aligned 3′ to 5′ at the replication fork, it may be continually duplicated by DNA polymerase, which expands the new DNA chain in the 5′ to 3′ direction.
- The new DNA chain with orientation 5′ to 3′ in the same direction as the migration of the fork is known as the leading strand. It extends from the replication origin.
- At the replication fork, the other template strand is orientated 5′ to 3′; hence, replicating it results in synthesis in the 3′ to 5′ direction, relative to the movement of the replication fork.
- This new DNA chain is referred to as the lagging strand and is synthesised discontinuously in the 3′ to 5′ direction. The DNA ligase then fuses these DNA fragments together to form a continuous DNA strand.
- As the leading strand is synthesised continuously and the lagging strand is synthesised discontinuously, the entire process is termed semi-discontinuous.
Formation of Okazaki Fragments
- The Okazaki fragments are made on the lagging strand when the DNA polymerase makes a part and then has to wait for the helicase to open up more of the DNA helix upstream.
- After the helicase opens up the DNA, the primase comes in and puts down a new complementary RNA primer. This lets the DNA polymerase join the DNA together and make the new Okazaki fragment.
- Most of the time, DNA is the material that holds the genes. The DNA is made up of two strands that run in opposite directions and are held together by hydrogen bonds.
- When a cell divides, all of the DNA in the genome needs to be copied. This doubles the amount of DNA in the first cell.
- In a semi-conservative mode, DNA copies itself so that one of the two strands in the new, double-stranded DNA is the original, or parent, strand.
- Because of this, both strands must act as models when DNA is copied. DNA polymerases are enzymes that help DNA copy itself.
- They can only make DNA that goes from 5′ to 3′. But because double-stranded DNA is not parallel, the process of making DNA must happen in either direction. So, the pieces come together when the lagging template strand is being made.
- Most of the time, the DNA polymerase adds nucleotides from the 5′ end to the 3′ end.
- The enzymes can add nucleotides to the growing strand on the leading strand over and over again.
- But because the strand runs from the 5′ end to the 3′ end, the growth of the chain of the newly made strand of DNA stops when it gets to the 5′ end. The replication fork is where another piece of DNA is made.
- This fork is where the unwinding of the double-stranded DNA starts, which is important for making new DNA strands from the parent strands.
- After the replication fork gets close to the double-strand, the DNA polymerase can join the nucleotides on the lagging strand.
- But the synthesis stops when it gets to the 5′ end of the RNA primer of the already synthesised stretch of DNA. Because of this, DNA synthesis at the lagging strand isn’t a continuous process. The stretches of DNA that are made are called Okazaki fragments.
Okazaki Fragments Function
- Okazaki fragments are small pieces of DNA that are made when the lagging strand is made without a break during DNA replication. It is important because it helps make the two daughter strands that are needed for a cell to divide.
- Okazaki fragments let the DNA polymerase make the missing strands in the segments, since it is not in the right place to make continuous strands.
- The main job of Okazaki fragments is to help the DNA polymerase make the lagging strand, even though it goes in the opposite direction. DNA polymerase I comes along and takes out the RNA primers and puts in DNA in their place. Once replication happens, the Okazaki pieces should be joined together to make one long strand. The DNA ligase that seals the sugar-phosphate backbone of the Okazaki fragments makes this possible. This makes it possible for two identical, continuous strands of daughter DNA to be made.
- Before a cell divides, the DNA must be copied. When a parent cell divides into two daughter cells, the DNA must be copied so that both daughter cells get the same genetic material. Cell division in unicellular organisms could be a way for them to reproduce asexually. Cell division in multicellular organisms, on the other hand, is important for the organism’s repair, growth, and for making the cells it needs for sexual reproduction.
Enzymes involved in Okazaki fragments formation
- Primase adds RNA primers to the trailing strand.
- This lets Okazaki fragments be made from 5′ to 3′. But primase makes RNA primers at a much slower rate than DNA polymerase does when making DNA on the leading strand.
- DNA polymerase on the lagging strand also has to be continually recycled to construct Okazaki fragments following RNA primers.
- Because of this, the lagging strand makes new DNA much more slowly than the leading strand.
- To fix this, primase acts as a short-term stop signal, stopping the replication fork for a short time during DNA replication.
- This process at the molecular level keeps the leading strand from catching up to the trailing strand.
2. DNA polymerase δ
- During this phase, enzymes that make DNA from the 5′ end to the 3′ end make new DNA.
- DNA synthesis needs DNA polymerase to make both the leading strand, which is made in one long piece, and the lagging strand, which is made in small pieces.
- This is done to make the newly made fragment bigger and to get rid of the RNA and DNA segment.
- Synthesis happens in three steps, and DNA polymerase α-primase and DNA polymerase δ are the two polymerases involved. This process starts when the clamp loader replication moves the polymerase -primase away from the RNA and DNA primer.
- This effect is what makes the clamp slide onto the DNA. After this, DNA polymerase δ begins to go into its holoenzyme form which then synthesis begins.
- The process of synthesis will keep going until the 5’end of the last Okazaki fragment arrives. Once it gets there, Okazaki fragment processing joins the newly made fragment to the strand that is still behind.
- The last thing that DNA polymerase δ does is help FEN1/RAD27 5′ Flap Endonuclease do its job. The rad27-p allele is lethal in most combinations but was viable with the rad27-p polymerase and exo1.
- Strong increases in CAN 1 duplication mutations are shown by both rad27-p polymerase and exo1.
- Only the double-strand break repair genes RAD50, RAD51, and RAD52 make this mutation possible. The RAD27/FEN1 makes nicks between Okazaki fragments that are next to each other by reducing the amount of strand-expulsion in the lagging strand.
3. DNA ligase I
- During lagging strand synthesis, DNA ligase I joins the Okazaki fragments together after DNA polymerase has changed the RNA primers to DNA nucleotides.
- If Okazaki fragments are not joined together, they could cause double-strand breaks, which split the DNA.
- Since the cell can only handle a small number of double-strand breaks and can only fix a small number of them, if enough ligation attempts fail, the cell could die.
- More research shows that proliferating cell nuclear antigen (PCNA) plays a role in addition to DNA ligase I’s job of joining Okazaki fragments.
- When the PCNA binding site on DNA ligase I is not working, it is much harder for DNA ligase I to connect Okazaki fragments.
- So, a proposed mechanism is that the DNA polymerase is released after a complex of PCNA and DNA polymerase makes Okazaki fragments.
- Then, DNA ligase I binds to the PCNA, which is clamped to the nicks in the lagging strand, and speeds up the formation of phosphodiester bonds.
4. Flap endonuclease 1
- Flap endonuclease 1 (FEN1) is in charge of putting Okazaki fragments together. It works with DNA polymerase to remove the RNA primer of an Okazaki fragment and can remove the 5′ ribonucleotide and 5′ flaps during lagging strand synthesis when DNA polymerase displaces the strands.
- When these flaps are taken off, a process called “nick translation” makes a “nick” that can be tied.
- So, Okazaki fragment maturation can’t happen without FEN1’s job of making a long, continuous DNA strand. During DNA base repair, the broken nucleotide is moved into a flap and then taken away by FEN1.
5. Dna2 endonuclease
- Dna2 endonuclease doesn’t have a specific structure, and its properties aren’t well known. However, it could be thought of as single-stranded DNA with free ends (ssDNA).
- During the Okazaki Process, you need Dna2 endonuclease to cut the long DNA flaps that leave FEN1.
- On Okazaki Fragments, the initiator RNA segment is taken out by the Dna2 endonuclease. Also, Dna2 endonuclease is a key player in the intermediates that are made when different types of DNA are broken down, and it helps keep telomeres in good shape.
- When a terminal RNA segment is attached at the 5′ end, Dna2 endonuclease becomes active because it moves from the 5′ end to the 3′ end. When a single-stranded DNA-binding protein RPA is present, the DNA 5′ flaps get too long, and the nicks no longer work as substrates for FEN1.
- This makes it impossible for the FEN1 to take off the 5′-flaps. So, Dna2’s job is to shorten the 3′ end of these fragments. This lets FEN1 cut the flaps and makes the maturation of the Okazaki fragments work better.
- During the Okazaki Process, there is no way to separate Dna2 helicase and endonuclease. The activity of Dna2 Endonuclease is not dependent on the 5′-tailed fork structure.
- It is known that unproductive binding can make it hard for FEN1 to cut and track. It is known that ATP slows down activity but helps the 3′-end label get released.
- Studies have shown that Okazaki fragment maturation is partly caused by a new model of Dna2 Endonuclease and FEN1.
Okazaki fragments are the DNA fragments that result from the discontinuous replication of the lagging strand.
Okazaki fragments are short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication.
DNA ligases are best known for their role in joining adjacent Okazaki fragments at the lagging strand of the replication fork; however, they are essentially involved in any process that requires sealing of phosphodiester bonds from the DNA backbone.
Primase. Primase adds RNA primers onto the lagging strand, which allows synthesis of Okazaki fragments from 5′ to 3′.
DNA ligase is the protein responsible for linking, or ligating, Okazaki fragments together in order to form a single complete DNA strand.
DNA ligase enzyme joins two Okazaki fragments together via phosphodiester bond between 3′ hydroxyl at the end of one fragment and a 5′ phosphate at the end of another fragment.
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