Replication is a process of DNA synthesis or DNA copying. It follows the semiconservative type of replication. A semiconservative replication results into two double-stranded DNA molecules. Each DNA has one strand retained from the parent and one newly synthesized strand. Various experiments tried revealing the mechanisms of replication. The process of replication in prokaryotes is clear in E. coli bacteria.

The process of DNA replication involves three steps such as initiation, elongation, and termination. The DNA replication requires replication enzymes and certain proteins. Arthur Kornberg and his colleagues worked on bacterial DNA replication and identified the DNA replication enzymes. They studied replication in E. coli.





Initiation of replication:

A DNA sequence known as the replicator directs the initiation of replication. This replicator includes the origin of replication. The origin of replication is known as Ori C. It is a specific region where the DNA denaturation occurs. Thus, it is a point at which the loss of native conformation of DNA results into the single-stranded structure, making the process of replication easier. The local DNA denaturation results in DNA replication bubble, leading to segments of single strands known as the template strands. The Y-shaped replication fork arises due to the exposure of two template strands. At this point, the two DNA strands get separated. A sequentially replicating segment of DNA is known as a replicon. The bacterial chromosomes contain a single replicator. The replication fork moves in the direction of untwisting of the DNA. There arise two replication forks while untwisting. Hence, the process of DNA replication is bidirectional. The Ori C is an AT-rich region. It gets denatured very easily.

An initiator DNA-a protein binds to the replicator. Thus, the DNA replication gets initiated due to stimulation of the denaturation process. The DNA helicase starts untwisting the DNA bi-directionally. It leads to the release of energy by hydrolysis of ATP. The energy gets utilized for untwisting. Then comes the role of DNA primases. The enzyme DNA primase forms a primosome complex. This enzyme synthesizes a short RNA primer. New nucleotides get added to the primer. At each replication fork, 10 to 100 nucleotides or pairs get added per second. The single strands created by the unwinding of DNA get stabilized by single-strand binding protein ( SSB ). Since there are two strands, one at the top and one at the bottom, the synthesis of RNA primer occurs on both the templates. The RNA primers get further lengthened by DNA polymerase III.





Okazaki fragments:

The DNA strands get synthesized from 5’ to 3’ direction at the Y-shaped replication fork. Two types of strands get synthesized. Leading strand is known as a continuous strand. A discontinuous strand is known as a lagging strand. The lagging strand gets synthesized in the form of discontinuous fragments known as Okazaki fragments.

Since the leading strand gets synthesized continuously, and the lagging strand is discontinuous, the process of replication is known as a semi-discontinuous type of replication. The two Okazaki strands get joined by DNA ligase enzyme.













Image: DNA Replication steps: The image describes five main steps of DNA replication in prokaryotes. (1)The unwinding of the DNA occurs due to helicase activity. The replication fork proceeds further. Two types of strands form including the leading strand and the lagging strand. The SSBPs, primase, and the DNA polymerase-III get recruited. These components allow new DNA synthesis. (2) In this step, the DNA polymerase-III get dissociated. Discontinuous strand synthesis starts on the lagging strands. These strands occur in the form of fragments. Thus, they are known as Okazaki fragments. (3) The replication fork gets extended little more. Thus, the discontinuous strand synthesis proceeds further. (4) The DNA polymerase-I replaces the RNA primer. (5) DNA ligase seals the gaps.





The gist of events occurring at the replication fork:

1. DNA primase synthesizes an RNA primer.

2. Replication of the lagging strand occurs.

3. Untwisting and elongation of new DNA strands.

4. Primer removal by DNA polymerase I.

5. Ligation of the adjacent fragments.





Circular DNA replicates bidirectionally:

The E. coli parental strand remains circular. These circular forms exhibit theta shaped structures. These structures arise as replication bubbles. As the two DNA strands untwist, the positive supercoils arise. Hence, the rotation helps in moving the molecule ahead. Topoisomerase solves the problem of supercoiling by introducing the negative supercoils. It keeps both the parent strands intact during replication. Hence, the unreplicated part undergoes a repeated negative supercoiling.





Rolling circle model:

It efficiently synthesizes multiple circular genome copies. Lambda bacteriophage uses this type of replication. A nick gets initiated at one of the parent strands. Then the strand rolls of a linear copy of the circular genome. The synthesis of the second strand converts the linear single-stranded genome into a double-stranded DNA.





References:

[1] IGenetics, Peter Russell, second edition

[2] DNA Replication, Arthur Kornberg, ‎Tania A. Baker, 2005

[3] The Cell Cycle: Principles of Control, Page 61, David Morgan, ‎David Owen Morgan, 2007



