Biosynthesis of DNA
The process by which the new double helical DNA synthesized from the existing DNA is called as "Replication". The mechanism of biosynthesis of DNA has been largely clarified by the discovery of the enzyme DNA Polymerase or DNA Nucleotidyl transferase. This enzyme catalyses the polymerization of mononucleotides to polynucleotides, which needs the following for its action.
1. A template strand dictates the synthesis of the new daughter strand and sequence of the template strand determines the addition of the nucleotides.
2. RNA primer to which subsequent nucleotides can be added.
3. Four nucleoside triphosphates namely, dGTP, dATP, dTTP and dCTP.
4. Magnesium ions as co-factor.
The energy required for this reaction is
provided by the hydrolysis of high energy bonds in the linear triphosphate
units of the dATP, dCTP, dGTP & dTTP. As each monomer is incorporated into
new chain, it loses its terminal pyrophosphate unit (PPi).
It is a process in which DNA copies itself to produce identical daughter molecules of DNA.
Three models of replication had been proposed. They are 1. Conservative replication 2.Dispersive replication and 3.Semi conservative replication.
According to this model, the parental DNA is conserved to one daughter cell and the newly synthesized DNA to another daughter cell.
According to this model, the parental DNA is unequally distributed (randomly) to the daughter cells.
Semiconseravative model of replication showing the daughter DNA having one parental strand and one daughter strand.
This model was established to be the correct mode of replication by the experiment carried out by Messlson and Stahl in 1957. E. coli cells were grown in a medium containing 15NH4Cl, for many generations such that the E.coli cells have density labelled 15N atoms in their DNA. The cells were then grown in a medium containing unlabelled NH4Cl. DNA was harvested from the daughter cells after one generation and the density of the DNA was analyzed using Cesium chloride density gradient centrifugation. If replication happens to occur by conservative model, two bands are corresponding to heavy DNA and other unlabelled DNA should be got. But if replication is semi-conservative, daughter DNAs should possess one labelled strand and one unlabelled strand, so they will form only one band with intermediate density. The results show that they form bands with intermediate density alone in the first generation, which confirms the semiconservative model of replication.
o Initiation of DNA synthesis occurs at a site called origin of replication.
o In prokaryotes, only one origin.
o In eukaryotes, there are multiple origins.
o The origin consists of a short sequence of A = T base pairs.
The two complementary strands of DNA separate at a site of replication to form a bubble. In eukaryotes many replication bubbles occur.
After the initiation point is located the unwinding of the DNA takes place once in every 10 nucleotide pairs. This allows the “strand separation”.
In prokaryotes the unwinding of the structure occurs with the help of an enzyme called helicase, which requires energy of hydrolysis of two ATP molecules per base pair broken. Another protein single strand binding protein (SSB), binds to the unwound DNA to prevent (rewinding) rejoining.
As the DNA polymerase cannot initiate replication a primer is a needed and the primer is the small nucleotide of RNA, synthesised by enzymes primase.
Under the influence of DNA polymerase, in the presence of Mg2+, the double strands of the DNA acting as a template (or primer) separate by cleaving the hydrogen bonds between complementary bases. The deoxyribo nucleoside triphosphates are attracted from solution in the cellular sap to form hydrogen bonds with their complementary bases on the separated strands of the (primer RNA) template dictates the sequence in which the monomers are assembled.
During this reaction, each incoming nucleotide loses a pyrophosphate group and forms an ester linkage with the 3' hydroxyl group of the deoxyribose on the existing last nucleotide. This linkage is called “phosphodiester linkage”.
The parental strands run in antiparallel direction. Synthesis occur simultaneously on both strands, but at different rates. No enzymes can synthesize 3' ® 5' direction and a single enzyme can not synthesize both strands. The single enzyme replicates one strand called leading strand in a continuous manner in 5' to 3' direction (forward), it replicates the other strand, lagging strand in a discontinuous manner and polymerising only few (250) nucleotides again run in 5' to 3' at backward direction. This is called semicontinuous DNA synthesis. The newly synthesized DNA is made as discontinuous small fragments called as Okazaki fragments and joined by the enzyme called ligase.
Thus 2 daughter double helices are formed each consisting of an old strand of the primer DNA and a complementary new strand. The final composition and nucleotide sequence of each strand is identical with the corresponding strand in the template (parent) DNA. This process has been named as replication.