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DNA Replication in Prokaryotes


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I write this article to explain the mechanism of DNA replication in prokaryotes. DNA replication is a vital part of cell division and this article explains the enzymes involved, the methods used and the theory behind prokaryotic DNA replication.



DNA Replication


The twentieth century saw great advances in the field of molecular biology. Discoveries that shook the very foundations of biology were made, and these discoveries opened up a new area of research in molecular biology. These discoveries began with that of Griffiths when he proved that the DNA is the genetic material, then Watson and Crick followed by discovering the double helical structure of DNA. However, with this data in mind, the question then turned to the mechanism of DNA inheritance. Since DNA is the genetic material in cells, and all living cells contain DNA, then during cellular division, the DNA must be duplicated so that each of the daughter cells can receive the genetic information from the parent cell. This process is called DNA replication and the mechanism of DNA replication was discovered by Arthur Kornberg for which he received the Nobel Prize in 1959.
However, before Kornberg discovered the mechanism of DNA replication, there was the question of the nature of process. There were two possibilities. One, that the replication is conservative in nature, implying that the parent DNA strand remains intact, and the intact strand is copied right from scratch and a new DNA molecule is formed. The other possibility is that DNA replication is semi-conservative in nature. The semi-conservative model of DNA replication stated that the parent DNA strand unwinds to form two strands, and each of the two strands is used as a template for the synthesis of the two daughter strands. Thus, each of the daughter cell would receive one DNA molecule containing one strand from the parent and one newly synthesized strand. The semi-conservative model of DNA replication was proved by the Mathew Messelson and Franklin Stahl in what is dubbed as "the most beautiful experiment in biology".

The most beautiful experiment in biology


The Messelson-Stahl experiment, involved culturing of Escherichia coli bacteria, on a medium containing "heavy" nitrogen (N15). After one generation, the culture was subjected to density gradient centrifugation, and the result was that the culture had deposited at the very bottom of the vial, indicating that all the nitrogen in the DNA was "heavy". This culture was then further sub-cultured in a medium containing "light" nitrogen (N14). After one generation, when density gradient centrifugation was performed, the resulting band was in the middle of the vial, indicating that the nitrogen in the DNA was a mixture of "heavy" and "light" strains. Upon a second sub-culture into a second medium, again containing "light" nitrogen, the density gradient centrifugation result indicated the presence of a strong band at the very top of the vial, and a lighter band in the middle, thus proving beyond doubt that the mechanism of DNA replication was semi-conservative in nature.

Mechanism of semi-conservative DNA replication


After Messelson and Stahl's experiment established the nature of DNA replication, Arthur Kornberg set out to explain the mechanism behind the process. He found that, DNA replication occurs during the S phase of the cell cycle. Enzymes and other proteins with highly specific functions take part in the process, whose exact mechanism is as described below.
The parent DNA molecule is unwound by the action of certain specific enzymes called DNA helicases. These bind to a point on the DNA molecule and begin unwinding the DNA double helix into two single strands. An enzyme called DNA gyrase (also called topo-isomerase I), helps to unwind the supertwists in the helix. Specialized proteins called SSB (Single Strand Binding) proteins then act upon the two separated strands and stabilize them. The primary enzyme that adds nucleotides to the parent strand is called DNA polymerase. However, it has two limitations. One, that it can only add nucleotides to an existing strand, and two, that it can only add nucleotides in the 5'-3' direction. Due to the second drawback, DNA replication occurs separately in the two strands. In the 5'-3' strand, it occurs normally as one continuous stretch and it is called the leading strand. However, in the other strand, synthesis occurs in bits and pieces, which are later joined together. The first drawback mentioned above is overcome by the action of an enzyme called RNA primase, which synthesizes an RNA primer (a short stretch of nucleotides, about 100bp in length), complimentary to the parent strand. The DNA polymerase enzyme adds nucleotides complimentary to those present in the parent leading strand and this continues until the end of the molecule is reached. In the lagging strand, the RNA primase keeps synthesizing RNA primers, and DNA polymerase adds DNA nucleotides in the 5'-3' direction in the form of short fragments called Okazaki fragments, after Reiji Okazaki, the scientist who discovered them. The DNA polymerase keeps adding nucleotides until the end of the previous Okazaki fragment is reached, after which a new primer is formed again. Once the fragments are synthesized, an enzyme called DNA ligase joins all the Okazaki fragments together to form one complete DNA molecule.
These newly synthesized DNA molecules are then separated and they are inherited by the daughter cells. The mechanism of this DNA replication is even more complex in eukaryotes with a lot more enzymes, and proteins participating in it. Another difference between the eukaryotic and prokaryotic replication is that the eukaryotic DNA Is complexed with histone proteins, which are absent in prokaryotes.

A new discovery to a new world of possibilities


The discovery of this mechanism, threw open a wide variety of fields for research. Scientists working on Genetic Engineering had the usage of enzymes such as DNA ligase at their disposal. The microarray technology and Polymerase Chain Reaction (PCR) are built on foundation stone of the DNA polymerase enzyme, and scientists working to cure diseases had a new approach towards controlling microbial growth-by inhibiting the action of the enzymes involved in DNA replication. DNA replication is indeed, a beautiful process, whose discovery showed us the way to a much more beautiful world of new possibilities, and approaches in science, towards making a better tomorrow.
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