Okazaki fragments are short sequences of DNA nucleotides (approximately 150 to 200 base pairs long in eukaryotes) which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication. They were discovered in the 1960s by husband and wife Reiji and Tsuneko Okazaki, along with the help of some of their colleagues.
During DNA replication, the double helix is unwound and the complementary strands are separated by the enzyme DNA helicase, creating what is known as the DNA replication fork. Following this fork, DNA primase and DNA polymerase begin to act in order to create a new complementary strand. Because these enzymes can only work in the 5' to 3' direction, the two unwound template strands are replicated in different ways. One strand, the leading strand, undergoes a continuous replication process since its template strand has 3' to 5' directionality, allowing the polymerase assembling the leading strand to follow the replication fork without interruption. The lagging strand, however, cannot be created in a continuous fashion because its template strand has 5' to 3' directionality, which means the polymerase must work backwards from the replication fork. This causes periodic breaks in the process of creating the lagging strand. The primase and polymerase move in the opposite direction of the fork, so the enzymes must repeatedly stop and start again while the DNA helicase breaks the strands apart. Once the fragments are made, DNA ligase connects them into a single, continuous strand. The entire replication process is considered "semi-discontinuous" since one of the new strands is formed continuously and the other is not.
During the 1960s, Reiji and Tsuneko Okazaki conducted experiments involving DNA replication in the bacterium Escherichia coli. Before this time, it was commonly thought that replication was a continuous process for both strands, but the discoveries involving E. coli led to a new model of replication. The scientists found there was a discontinuous replication process by pulse-labeling DNA and observing changes that pointed to non-contiguous replication.
Experiments
The work of Kiwako Sakabe, Reiji Okazaki and Tsuneko Okazaki provided experimental evidence supporting the hypothesis that DNA replication is a discontinuous process. Previously, it was commonly accepted that replication was continuous in both the 3' to 5' and 5' to 3' directions. 3' and 5' are specifically numbered carbons on the deoxyribose ring in nucleic acids, and refer to the orientation or directionality of a strand. In 1967, Tsuneko Okazaki and Toru Ogawa suggested that there is no found mechanism that showed continuous replication in the 3' to 5' direction, only 5' to 3' using DNA polymerase, a replication enzyme. The team hypothesized that if discontinuous replication was used, short strands of DNA, synthesized at the replicating point, could be attached in the 5' to 3' direction to the older strand.
To distinguish the method of replication used by DNA experimentally, the team pulse-labeled newly replicated areas of Escherichia coli chromosomes, denatured, and extracted the DNA. A large number of radioactive short units meant that the replication method was likely discontinuous. The hypothesis was further supported by the discovery of polynucleotide ligase, an enzyme that links short DNA strands together.
In 1968, Reiji and Tsuneko Okazaki gathered additional evidence of nascent DNA strands. They hypothesized that if discontinuous replication, involving short DNA chains linked together by polynucleotide ligase, is the mechanism used in DNA synthesis, then "newly synthesized short DNA chains would accumulate in the cell under conditions where the function of ligase is temporarily impaired." E. coli were infected with bacteriophage T4 that produce temperature-sensitive polynucleotide ligase. The cells infected with the T4 phages accumulated a large number of short, newly synthesized DNA chains, as predicted in the hypothesis, when exposed to high temperatures. This experiment further supported the Okazakis' hypothesis of discontinuous replication and linkage by polynucleotide ligase. It disproved the notion that short chains were produced during the extraction process as well.
The Okazakis' experiments provided extensive information on the replication process of DNA and the existence of short, newly synthesized DNA chains that later became known as Okazaki fragments.
Pathways
Two pathways have been proposed to process Okazaki fragments: the short flap pathway and the long flap pathway.
Short Flap Pathway
In the short flap pathway in eukaryotes the lagging strand of DNA is primed in short intervals. In the short pathway only, the nuclease FEN1 is involved. Pol δ frequently encounters the downstream primed Okazaki fragment and displaces the RNA/DNA initiator primer into a 5′ flap. The FEN1 5'-3' endonuclease recognizes that the 5' flap is displaced, and it cleaves, creating a substrate for ligation. In this method the Pol a-synthesized primer is removed. Studies show that in the FEN1 suggest a 'tracking; model where the nuclease moves from the 5' flap to its base to preform cleavage. The Pol δ does not process a nuclease activity to cleave the displaced flap. The FEN1 cleaves the short flap immediately after they form. The cleavage is inhibited when the 5' end of the DNA flap is blocked either with a complementary primer or a biotin-conjugated streptavidin moiety. DNA ligase seals the nick made by the FEN1 and it creates a functional continuous double strand of DNA. PCNA simulates enzymatic functions of proteins for both FEN1 and DNA ligase. The interaction is crucial in creating proper ligation of the lagging DNA strand. Sequential strand displacement and cleavage by Pol δ and FEN1, respectively, helps to remove the entire initiator RNA before ligation. Many displacements need to take place and cleavage reactions are required to remove the initiator primer. The flap that is created and processes and it is matured by the short flap pathway.
Long Flap Pathway
In some cases, the FEN1 lasts for only a short period of time and disengages from the replication complex. This causes a delay in the cleavage that the flaps displaced by Pol δ become long. When the RPA reaches a long enough length, it can bind stably. When the RPA bound flaps are refactorized to FEN1 cleavage the require another nuclease for processing, this has been identified as an alternate nuclease, DNA2. DNA2 has defects in the DEN1 overexpression. The DNA2 showed to work with FEN1 to process long flaps. DNA2 can dissociate the RPA from a long flap, it does this by using a mechanism like the FEN1. It binds the flap and threads the 5' end of the flap. The nuclease cleaves the flap making it too short to bind to the RPA, the flap being too short means it is available for FEN1 and ligation. This is known as the long flap method. DNA2 can act as FEN1 as a backup for nuclease activity but it is not an efficient process.
Alternate pathway
Until recently, there were only two known pathways to process Okazaki fragments. However, current investigations have concluded that a new pathway for Okazaki fragmentation and DNA replication exists. This alternate pathway involves the enzymes Pol δ with Pif1 which perform the same flap removal process as Pol δ and FEN1.
Enzymes involved in fragment formation
Primase
Primase adds RNA primers onto the lagging strand, which allows synthesis of Okazaki fragments from 5' to 3'. However, primase creates RNA primers at a much lower rate than that at which DNA polymerase synthesizes DNA on the leading strand. DNA polymerase on the lagging strand also has to be continually recycled to construct Okazaki fragments following RNA primers. This makes the speed of lagging strand synthesis much lower than that of the leading strand. To solve this, primase acts as a temporary stop signal, briefly halting the progression of the replication fork during DNA replication. This molecular process prevents the leading strand from overtaking the lagging strand.
DNA polymerase δ
New DNA is made during this phase by enzymes which synthesize DNA in the 5' to 3' direction. DNA polymerase is essential for both the leading strand which is made as a continuous strand and lagging strand which is made in small pieces in DNA Synthesis. This process happens for extension of the newly synthesized fragment and expulsion of the RNA and DNA segment. Synthesis occurs in 3 phases with two different polymerases, DNA polymerase α-primase and DNA polymerase δ. This process starts with polymerase α-primase displacing from the RNA and DNA primer by the clamp loader replication Effect, this Effect leads the sliding clamp onto the DNA. After this, DNA polymerase δ begins to go into its holoenzyme form which then synthesis begins. The synthesis process will continue until the 5'end of the previous Okazaki fragment has arrived. Once arrived, Okazaki fragment processing proceeds to join the newly synthesized fragment to the lagging strand. Last function of DNA polymerase δ is to serve as a supplement to FEN1/RAD27 5' Flap Endonuclease activity. The rad27-p allele is lethal in most combinations but was viable with the rad27-p polymerase and exo1. Both rad27-p polymerase and exo1 portray strong synergistic increases in CAN 1 duplication mutations. The only reason this mutation is viable is due to the double-strand break repair genes RAD50, RAD51 and RAD52. The RAD27/FEN1 creates nicks between adjacent Okazaki fragments by minimizing the amount of strand-expulsion in the lagging strand.
DNA ligase I
During lagging strand synthesis, DNA ligase I connects the Okazaki fragments, following replacement of the RNA primers with DNA nucleotides by DNA polymerase δ. Okazaki fragments that are not ligated could cause double-strand-breaks, which cleaves the DNA. Since only a small number of double-strand breaks are tolerated, and only a small number can be repaired, enough ligation failures could be lethal to the cell.
Further research implicates the supplementary role of proliferating cell nuclear antigen (PCNA) to DNA ligase I's function of joining Okazaki fragments. When the PCNA binding site on DNA ligase I is inactive, DNA ligase I's ability to connect Okazaki fragments is severely impaired. Thus, a proposed mechanism follows: after a PCNA-DNA polymerase δ complex synthesizes Okazaki fragments, the DNA polymerase δ is released. Then, DNA ligase I binds to the PCNA, which is clamped to the nicks of the lagging strand, and catalyzes the formation of phosphodiester bonds.
Flap endonuclease 1
Flap endonuclease 1 (FEN1) is responsible for processing Okazaki fragments. It works with DNA polymerase to remove the RNA primer of an Okazaki fragment and can remove the 5' ribonucleotide and 5' flaps when DNA polymerase displaces the strands during lagging strand synthesis. The removal of these flaps involves a process called nick translation and creates a nick for ligation. Thus, FEN1's function is necessary to Okazaki fragment maturation in forming a long continuous DNA strand. Likewise, during DNA base repair, the damaged nucleotide is displaced into a flap and subsequently removed by FEN1.
Dna2 endonuclease
Dna2 endonuclease does not have a specific structure and their properties are not well characterized, but could be referred as single-stranded DNA with free ends (ssDNA). Dna2 endonuclease is essential to cleave long DNA flaps that leave FEN1 during the Okazaki Process. Dna2 endonuclease is responsible for the removal of the initiator RNA segment on Okazaki Fragments. Also, Dna2 endonuclease has a pivotal role in the intermediates created during diverse DNA metabolisms and is functional in telomere maintenance.