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What are the Okazaki fragments? How are they formed?

Understand the meaning of Okazaki fragments and know how they formed.

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Okazaki fragments

Okazaki fragments are defined as the short sequence of DNA nucleotides that are thought synthesized discontinuously, interconnected in future trough DNA ligase in order to develop lagging strand at the time of DNA replication. These are in its simplest form, are referred as the short, newly synthesized DNA fragments. These fragments are short, newly synthesized DNA fragments, which together can form short double-stranded DNA sections. Okazaki fragments are between 1,000 and 2,000 nucleotides long in Escherichia coli and are between 100 and 200 nucleotides long in eukaryotes.

They are separated by ~10-nucleotide RNA primers and are unligated until RNA primers are removed, followed by enzyme ligase connecting the two Okazaki fragments into one continuous newly synthesized complementary strand. On the leading strand DNA replication proceeds continuously along the DNA molecule as the parent double-stranded DNA is unwound, but on the lagging strand the new DNA is made in installments, which are later joined together by a DNA ligase enzyme.

The ability of DNA to work towards one direction is the fundamental reason, for which it happens. On the leading strand this route is continuous, but on the lagging strand it is discontinuous. They were originally discovered in 1966 by Kiwako Sakabe, Reiji Okazaki, and Tsuneko Okazaki in their research on DNA replication of Escherichia coli. They were further investigated by them and their colleagues through their research including the study on bacteriophage DNA replication in Escherichia coli.

The fundamental purpose of Okazaki fragments is to determine the position of DNA. On the strand of the template, which dictates new DNA synthesis away from the direction of replication fork movement. As DNA is anti-parallel and hold the ability to synthesise in one direction, such fragments are formed during DNA replication. Because of this, at each replication fork, there is a leading strand that is synthesized in the 3’ to 5’ direction and a lagging strand, synthesized in the 5' to 3' direction. Okazaki fragments form because the lagging strand that is being formed have to be formed in segments of 100–200 nucleotides. This is done DNA polymerase making small RNA primers along the lagging strand. These are produced Okazaki fragments form because the lagging strand that is being formed have to be formed in segments of 100–200 nucleotides. This is done DNA polymerase making small RNA primers along the lagging strand.

After the Okazaki fragments (sequences complementary to the lagging strand of DNA) are produced, they must be joined together to produce a continuous strand (Figure 1.12). On the lagging strand, DNA polymerase III of Escherichia coli stops when it reaches the RNA primer at the 5′ end of the next Okazaki fragment. Then, DNA polymerase I of E. coli removes the ribonucleotides from the Okazaki fragment and replaces them with deoxyribonucleotides. The reason, for which such fragments form are as follows:

  • To enable replication of the 3′ → 5′ (lagging) strand
  • To form the template for the RNA primers
  • To initiate replication on the 5′ → 3′ (leading) strand
  • To help the DNA helicase unwinding the DNA helix

The okazaki fragments formed during replication enables the replication of the 3’ 5’ (lagging strand). They are short sequences of DNA nucleotides newly synthesize on the lagging strand. It acts as the building block for the synthesis of DNA in the lagging strand. The fragments are synthesize discontinuously and are later linked together by enzyme DNA ligase to create the lagging strand during DNA replication.

During DNA replication the DNA duplex is unwound and two daughter strands are formed at the replication fork. DNA polymerase can only add nucleotides in the 5' to 3' direction, hence the synthesis of the one of the daughter strands, the leading strand, can proceed continuously in the same direction as movement of the replication fork. Still, in the entire process, complication arises with the replication of the other daughter strand, the lagging strand. As growth must occur in the 5' to 3' direction the copying of the template must somehow occur in the opposite direction as the movement of the replication fork. There are two different ways such as the short flap pathway and the long flap pathway, through which the Okazaki Fragments form.

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. Through this method, the Pol a-synthesized primer is removed. In some of the major cases, the okazaki fragments are formed through Long Flap Pathway. In this case, 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 require another nuclease for processing, this has been identified as an alternate nuclease, DNA2. DNA2 has defects in the DEN1 overexpression. 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.

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