This is the enzyme which polymerises deoxyribonucleotides. It adds deoxyribonucleotides to the 3’0II end of a growing polynucleotide. The new nucleotide comes as nucleoside-triphosphate and is joined to the open -Oil of polynucleotide by the removal of pyrophosphate.
Polynucleotide (n) + d XTP Polymerase
Polynucleotide (n+i) + PPi (Polymerase) Arthur Romberg and his colleagues in Washington University in 1956 isolated the first DNA polymerase from E.coli. It was then known as Romberg enzyme.
It was later named as DNA polymerase I due to the discoveries of other polymerases like Polymerase II and polymerase III from the same strain of E. coli. Polymerase III is replication. Plymerase I and II are involved in DNA repair and proof reading in prokaiyotes. DNA polymerase (Pol) requires a template for synthesis of a new strand.
They can synthesise only in the 5′-3′ direction. This enzyme cannot start DNA synthesis, rather can only add to an existing primer strand. A primer is a small DNA or DNA strand hydrogen bonded to the template. During DNA synthesis new nucleotides are added to the open 3′ OH end of the primer or growing polynucleotide so that the synthesis is always in the 5′-3′ direction only. Polymerase III has got exonuclease property so that it can remove nucleotides from the 3′ end of the growing DNA strand (3′-5′ exonuclease).
It helps in proof reading so that any wrong nucleotide added at 3′ end can be removed. Pol I has 5′-3′ exonuclease function so that it can remove short DNA primers from DNA-DNA hybrid.
Mechanism of DNA Replication
The entire set of enzymes and protein factors involved in DNA replication is known as rcplicase system or rcplisomc. The initiator protein recognizes the unique sequence of origin and bind to.it.DNA hclicasc enzyme unwinds the double stranded DNA by breaking the hydrogen bonds between the nitrogenous bases.
Then single strand binding proteins (SSB proteins) bind to the separated strands to keep them in extended position and also to prevent rewinding and attack by single straind nuclease. As a result of the combined action of the enzyme helicasc and the protein factors SSB a “V”-shaped fork is created at the origin known as replication fork. One must understand that in a bidirectional replication two replication forks are created in opposite direction at the origin.
The open ends of the two forks meet at the origin and appear like two “V”s facing each other. As the replication fork moves through the unwinding of the DNA strands a positive super coil is created in the unreplicated portion of DNA ahead of the fork. This is like a knot ahead of fork so that further movement of the fork is hindered.
This super coiling is removed by an enzyme called as topoisomerase II or Gyrase in E.coli and topoisomerase I in eukaryotes.In E.coli the enzyme makes cut on both the strands of the circular DNA and then one segment of DNA passes through other to relieve the super coil and then the cut is sealed. DNA polymerase requires a primer strand for the addition of nucleotides. Enzyme primase synthesizes a short primer complementary to the 3′ end of the templates.
The replication fork moves by unwinding the double stranded DNA.As a result, one template strand is continuous with the replication fork.ie, the direction of movement of fork is along the 3′ to 5′ direction of the template strand. In the same replication fork the other strand is not continuous with the movement of the fork as the fork opens behind the 3′ end of this template strand. The template strand whose 3′-5′ direction coincides with the movement of fork is known as leading template strand or leading strand. This strand requires a single initiation event at the start of the replication and then the new DNA synthesis takes place continuously.
The other strand whose 3′-5′ direction is opposite to the direction of the movement of replication fork is known as lagging strand. On the lagging strand the direction of DNA synthesis (always in 5′- 3′ direction) and movement of fork are in opposite direction. In this case, continous DNA synthesis is not possible rather short DNA strands are synthesized discontinuously which are later joined.
Synthesis of each strand coincides a single movement of the fork and necessitates an initiation event each. The small DNA strands on the lagging strand are called Okazaki fragments after the Japanese Scientist Reiji Okazaki who first observed those fragments. He observed fragments of 1000-2000 nucleotides long in prokaryotes and 100-200 nucleotides long in eukaryotes.
One must understand that in a bidirectional replication, from the origin of replication a particular template strand is leading strand along the movement of one replication fork and lagging strand along the opposite replication fork.
Each Okazaki fragment on the lagging strand has its own primer. I he primosome protein complex moves along the
lagging strand and forms RNA primer at intervals on which Okazaki fragments are synthesized. DNA polymerase 1 enzyme removes the DNA primers from the lagging strand through its 5′-3′ exonuclease activity and fills the resulting gaps by adding nucleotides complementary to those portions of lagging strand.
Finally Ligase enzyme joins the Okazaki fragments to give a continuous DNA strand complementary to lagging strand.