Semi-conservative replication of DNA

Replication: The process in which a DNA molecule produces exact copy or replica of itself.

Replication of DNA takes place in nucleus, during S-phase of Cell cycle.

It takes place in following steps:

1. Activation of Nucleotides
2. Origin or initiation point
3. Unwinding of DNA strand
4. Synthesis of New strand
5. Leading and Lagging strand
6. Formation of Daughter Molecules

1. Activation of Nucleotides:.

- 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.

- Activation of nucleotides (dAMP + ATP dATP + AMP) is done by enzyme phosphorylase.

2. Origin or initiation point:

- Replication begins at certain specific sites called as origin points or initiation points.

- In eukaryotes. there are several origin points.

- At the origin by formation of an incision (nick).

- At the nick, breaking of hydrogen bonds between the two strands initiated (unzipping).

3. Unwinding of DNA strand

- After incision by endonuclease enzyme, the DNA strands are unwinded.

- The unwinding of DNA strand is done by Helicase. Helicase = rep protein.

- 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.

4. Synthesis of New strand

- The separated strands act as "Template" or "Mould" for complementary strand.

- The synthesis of complementary strand begins with synthesis of small RNA.

- The small RNA is called as "RNA primer". It is synthesized by RNA Primase.

- The DNA polymerase puts new nucleotides on the 5' end of RNA primer.

- The synthesis of new strand takes place from 5' to 3'.

 

5. Leading and Lagging strand

• DNA - polymerase catalyses polymerization 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' to 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" to 3' direction, called Okazaki fragments.

• The Okazaki fragment 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.

• The gaps are filled by DNA polymerase I.

6. Formation of Daughter Molecules

• 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 ¹⁵N and E.coli

• In prokaryotcs circular DNA is present There is only one origin and the replication is called "theta' replication (Theta replication) as it looks like Greek - letter Theta.