The process in which a DNA molecule produces exact copy or replica of itself is known as replication of DNA. In eukaryotes, replication of DNA takes place inside nucleus.
Mechanism of DNA replication:
Activation of Nucleotides :
All the four types of DNA nucleotides are found in the nucleoplasm in the form of their monophosphates. They are activated into triphosphates like dATP, dGTP, dTTP and dCTP using ATP. It occurs in presence of the enzyme phosphorylase and the process is called activation of nucleotides. (dAMP + ATP → dATP + AMP)
Origin or Initiation point :
DNA replication begins at certain specific sites situated on the molecule. Such sites are called origin points or initiation points. In eukaryotes, there are several origin points. During replication, DNA molecule is split by the activity of enzyme at the origin by formation of an incision (nick). It initiates breaking of hydrogen bonds between the two strands (unzipping).
Unwinding of DNA strands :
After the activity of endonuclease enzyme the strands of DNA are free from each other but not separated. This separation of the strands is done by DNA-unwinding protein i.e. helicase enzyme (also called rep protein ). Now the DNA molecule appears as inverted 'Y'-shaped structure called replication fork. The separated strands are prevented from coiling by SSBP (Single strand DNA binding protein) or helix destabilizing protein.
Synthesis of new strands :
Now each separated strand acts as a template or mould for synthesis of a complementary new strand. It takes place with the help of small RNA molecule called RNA primer. Synthesis of this RNA primer is controlled by the enzyme RNA primase. It gets attached at the 3' end of the template strand. RNA primer attracts complementary nucleotides from the surrounding nucleoplasm. It occurs under the influence of the enzyme DNA polymerase. The new complementary nucleotides are arranged on this strand to build a strand in 5' → 3' direction.
Leading and lagging strands :
DNA - polymerase catalyses polymerisation only in one direction, that is 5' → 3'. Consequently, on one strand replication is continuous, while on the other strand it is discontinuous. The strand which opens from 3' → 5' is called leading template and its complementary strand is called leading strand. It is constructed continuously at a faster rate. The other strand which opens from 5' → 3' is called lagging template and its complementary strand is called lagging strand. It is constructed discontinuously at a slower rate. The lagging strand is constructed in the form of short fragments of DNA in 5' → 3' direction, called Okazaki fragments, which are later joined by the enzyme DNA ligase. Each Okazaki fragment needs one RNA primer. Later the RNA primer is removed by the enzyme RNAse.
Formation of daughter DNA molecules :
In this way for each old strand , a new complementary strand is constructed. Simultaneously both the strands (new and old) undergo coiling and two identical daughter DNA molecules are formed at the end of the process.
After replication each DNA molecule has one old and the other new strand. It shows that 50% part of the mother molecule is retained or conserved while remaining 50% part is newly constructed. Hence the process is referred to as semi-conservative replication.
Meselson and Stahl (1958) experimentally proved semi-conservative nature of DNA replication using heavy isotope of nitrogen N and E.coli.
In prokaryotic circular DNA is present. There is only one origin and the replication is called 'θ' replication. (Thera replication) as it looks like Greek - letter Theta.