Following steps take place in the replication of DNA:
- Origin of replication
The replication of a DNA molecule begins at special sites called origins of replication. The cukaryotic DNA contains thousand of such replication origins. A protein initiates DNA replication. It recognizes these sequences of origins and attach to the DNA. It separates the two strands. These strands open up to form a replication “bubble.” Multiple replication bubbles are formed in eukaryotes. These bubbles fuse with each other. The replication of DNA then proceeds in both directions and entire ‘molecule is copied. There is replication fork at each end of a replication bubble. It is Y‑shaped region. New strands of.DNA. elongates replication fork.
- Elongating a New DNA Strand
An enzymes DNA polymerases catalyzes elongation of new DNA at a replication fork. The nucleotides align with complementary basis on “old” template strand of DNA. They are added by DNA polymerase one by one. The rate of elongation is about 500 nucleotides per second in human cells
The substrates for DNA are nucleo side triphosphate. The nucleoside triphosphates have three phosphate groups like ATP. Each monomer loses two phosphates and joins to the growing end of a DNA strand. Hydrolysis of the phosphate is the exergonic reaction. Therefore it drives polymerization of nucleotides to form DNA.
- The Problem of Antiparallel DNA Strands
There is a problem of DNA synthesis at the replication fork. The two DNA strands are antiparallel (3-5 and 5-3). Their sugar—phosphate backbones run in opposite directions. Phosphate group of each nucleotide is attached to the 5′ carbon of deoxyribose. The phosphate group of one nucleotide is joined to the 3′ carbon of the adjacent nucleotide. Therefore, there is different mechanism of replication in both strands:
(a) Leading strand: The enzyme DNA polymerase can only add
nucleotides to the free 3′ end of a DNA strand. It can never add it to the 5′ end. Thus, a new DNA strand is formed in 5— 3′ directions. The DNA polymerase can synthesize a continuous complementary strand along 5′ —3 direction. This DNA strand is called the leading strand.
(b) Lagging strand: The DNA polymerase move away from the replication fork to elongate in 3-5 strand of DNA. The DNA synthesized in this direction is called lagging. The lagging strand is first synthesized as a series of segments. These pieces are called Okazaki fragments. These segments were discovered by Japanese scientist Okazaki. These fragments are about 100 to 200 nucleotides long in eukaryotes.
4. RNA Primer
There is another problem for DNA polymerase. It can only add a nucleotide to a polynucleotide that is already correctly paired with the complementary strand. This means that DNA polymerase cannot actually initiate synthesis of a DNA strand. Nucleotides must be added to the end of an already existing chain. This chain of nucleotides is called a primer. The primer is a short stretch of RNA. It is synthesized by another enzyme primase. It is about 10 nucleotides long in eukaryotes. Only one primer is required for the leading strand of new DNA. Each fragment must have separate primer in the lagging strand. An enzyme then replaces the RNA nucleotides of the primers with DNA. Another enzyme Ligase joins all the DNA fragments into a strand.
Protein Assisting the DNA replication
Following proteins assist in the synthesis of DNA:
- DNA polymerase
- Helicase: it is a protein. It causes untwisting the double helix of DNA.
- Single strand binding protein: It is attached to the separated strands of DNA and does not allow them to recoil.
The errors in the completed DNA molecule are only one in one billion nucleotides. These errors must be corrected. Some enzyme removes these errors.