DNA REPLICATION

RNA WORLD

RNA was the first genetic material. There are evidence to suggest that essential life processes, such as metabolism, translation, splicing etc. evolved around RNA. RNA use to act as a genetic material as well as a catalyst, there are some important biochemical reactions in living system that are catalysed by RNA catalysts and not by Protein enzymes (e.g. splicing) RNA being a catalyst was reactive and hence unstable. Therefore, DNA has evolved from RNA with chemical modification that makes it more stable. DNA being double stranded and having complementary strand further resists changes by evolving a process of repair. RNA is adapter, structural molecule and in some case catalytic. Thus RNA is better material for transmission of information. 

DNA REPLICATION

1. While proposing the double helical structure for DNA, Watson and Crick had immediately proposed a scheme for replication of DNA. To quote their original statement that is as follows.

2. "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material"           ( Watson and Crick, 1953).

 

3. The scheme suggested that the two strands would separte and act as a template for the synthesis of new complementary strands. After the completion of replication, each DNA molecule would have one parental and one newly synthesised strand. This scheme was termed as semiconservative DNA replication.

4. DNA capable of self duplication.

5. All living beings have the capacity to reproduce because of this characteristic of DNA.

6. DNA replication takes place in "S-phase" of the cell cycle. At the time of cell division, it divides in equal parts in the daughter cells.

SEMI CONSERVATIVE MODE OF DNA REPLICATION

Semi conservation mode of DNA replication was first proposed by Watson and Crick. Later on it was experimentally proved by Meselson and Stahl (1958) in E.coli and Taylor in Vicia faba (1958). To prove this method, Taylor used Radiotracer Technique in which Radioisotopes (tritiated thymidine = ) were used. Meselon and Stahl used heavy isotope of nitrogen-15.

Matthew Meselson and Franklin Stahl performed the following experiment in 1958:

1. They grew E.coli in a medium containing NH4CL ( N-15 is the heavy isotope of nitrogen) as the only nitrogen source for many generations. The result was that N-15 was incorporated into newly synthesised DNA (as well as other nitrogen containing compounds). This heavy DNA molecule could be distinguished form the normal DNA by centrifugation in as cesium chloride (CsCl) density gradient (please note that N-15 is not a radioactive isotope, and it can be separated from N-14 only based on densities).

2. Then they transferred the cells into a medium with normal NH4CL and took samples at various definite time intervals as the cells multiplied, and extracted the DNA that remained as double stranded helices. The various samples were separated independently on CsCl gradient to measure the densities of DNA.

3. Thus, the DNA that was extracted from the culture one generation after the transfer from N-15 to N-14 medium [that is after 20 minutes; E.coli divides in 20 minutes] had a hybrid or intermediate density. DNA extracted from the culture after another generation [that is after 40 minutes, 2 generation] was composed of equal amounts of this hybrid DNA and of 'light' DNA.

MECHANISM OF DNA REPLICATION

 The following steps are included in DNA replication:-

1. Unzipping (Unwinding):-

 

1. The separation of 2 chains of DNA is termed as unzipping. And it takes place due to the breaking of H-bond. The process of unzipping starts at a certain specific points which is termed as initiation point or origin of replication. In prokaryotes there occurs only one origin of replication but in eukaryotes there occur many origin or replication i.e unzipping starts at many points simultaneously. At the place of origin the enzyme responsible for unzipping (breaking the hydrogen bond) is Helicase (=Swivelase). In the process of unzipping Mg+2 act as cofactor.

2. SSB (Single stranded DNA binding protein) prevents the information of H-bonds.

3. Topoisomerase (in prokaryotes also called as DNA gyrase)  release the tension arises due to supercoiling. 

Note: The process of DNA replication takes a few minutes in prokaryotes and a few hours in Eukaryotes.

2. Formation of New chain:- 

 

1. To start the synthesis of new chain, special type of RNA is required which is termed as RNA primer. The formation of RNA primer is catalysed by an enzyme- RNA Polymerase (primase). Synthesis of RNA-Primer takes place in 5'-3' direction. After the formation of New chain, this RNA is removed. For the information of new chain Nucleotides are obtained from Nucleoplasm. In the Nucleoplasm, Nucleotides are present in the form of triphosphates like dATP, dGTP, dCTP, dTTP etc. 

2. During replication, the 2 phosphate groups of all nucleotides are separated. In this process energy is yeilded which is consumed in DNA replication.

3. Energetically replication is a very expensive process. Deoxyribonucleoside triphosphase serve dual purposes in addition to acting as substrates they provide energy for Polymerisation. 

4. The formation of New chain always takes place in 5'-3' direction. As a result of this, one chain of DNA is continuously formed and it is termed as Leading strand. The formation of second chain begins from the centre and not form the terminal points, so this chain is discontinuous and is made up of small segments called Okazaki fragments. This discontinuous chain is termed as Lagging strand. Ultimately all these segments joined together and a complete new chain if formed. 

5. The Okazaki fragments are joined together by an enzyme DNA Ligase.

6. The formation of New chains in catalysed by an enzyme DNA Polymerase. In prokaryotes it is of 3 types:

1. DNA-polymerase 1 :- This was discovered by KORNBERG (1957). So it is also called as 'Kornberg's enzyme'. KORNBERG also synthesized DNA first of all, in the laboratory. This enzyme functions as exonuclease. It separates RNA-Primer from DNA and also fills the gap. It is also known as DNA-repair enzyme.

2. DNA-polymerase 2 :-  It is least reactive in replication process. It is also helpful in DNA-repairing in absence of DNA-polymerase 1 and DNA-polymerase 3.

3. DNA-polymerase 3 :- This is the main enzyme in DNA-replication. It is most important. The larger chains are formed by this enzyme. This is also known as Replicase. 

7. In the semi conservative mode of replication each daughter DNA molecule receives one chain of polynucleotides from the mother DNA- molecule and the second chain is synthesized.

Sepcial points:-

1. All DNA polymerase 1,2 and 3 enzymes have 5'-3' Polymerisation activity and 3'-5' exonuclease activity.

2. DNA polymerase 1 also has 5'-3' exonuclease activity.

3. Any failure in cell division after DNA replication result into polyploidy.

4. Difference between DNAs and DNase is that DNAs means many DNA and DNase means DNA digestive enzymes.

 

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