DNA replication is a process by which DNA makes copies of itself. This is a fundamental process of all the living organisms having DNA as their carrier for genetic material. This process of DNA replication takes place in the S-phase (synthesis phase) of the cell cycle right before the cell divides into two daughter cells. The DNA content is required to be doubled in order to divide it equally into both the daughter cells.
DNA (deoxyribonucleic acid), as explained by Watson and Crick, is a double helix structure. It is composed of two strands of polynucleotides coiled with each other to form a helix. The two strands are joined with each other by a series of hydrogen bonds between the bases of both the strands.
The process of DNA replication is different in eukaryotes and prokaryotes due to many factors like different enzymes are involved, different start and stop points, the difference in the structure of the DNA etc. The main role in the process of DNA replication is played by the enzymes that catalyze this process. Interestingly, there are different enzymes that are assigned different roles for each step. Let us find out about these enzymes and their roles.
DNA Replication Enzymes in Prokaryotes
The main enzyme that works in DNA replication both in case of Prokaryotes and eukaryotes is DNA Polymerase, but there is structural and functional dissimilarity in both of the enzymes. DNA polymerase, as the name depicts, is responsible for adding new nucleotides (dNTPs) to the growing end of the DNA strand. There is a fixed pattern of bonding between the nucleotides which is followed while adding the nucleotides on the growing end by the DNA polymerases. This rule of base pairing is called as Chargoff’s rule. Therefore while adding the new nucleotide, the DNA polymerase strictly follows Chargoff’s rule of base pairing and thus successful leads to the formation of new strands from the old (template) strand.
Following are the 5 types of DNA Polymerase in Prokaryotes with their role;
DNA polymerase 1
This DNA polymerase has maximum activities which are listed below:
- It has 3′ to 5′ exonuclease activity which is used for proofreading the daughter strand for any error in the addition of the nucleotides. If any error is detected, the DNA synthesis is halted until the error is rectified by the action of other enzymes.
- It has 5′ to 3′ exonuclease activity which is used for primer removal. This is the exclusive activity of DNA pol 1.
- It has 5′ to 3′ polymerase activity which is used for the synthesis of the DNA from 5′ to 3′ end. This activity is common in all types of DNA polymerases.
- It also has gap filling and DNA repairing activity. This gap-filling activity is utilized in filling the gaps between Okazaki fragments.
DNA Polymerase 2
- It has 5′ to 3′ polymerase activity.
- It has 3′ to 5′ exonuclease activity (proofreading)
- It has DNA Repair activity.
DNA Polymerase 3
This is the main replicating enzymes in prokaryotes which is mainly responsible for the synthesis of new DNA strands (5′ to 3′ polymerase activity) by bringing in new nucleotides.
It is composed of a complex structure with alpha, epsilon and theta subunits in its core. These 3 subunits are responsible for following main functions:
- DNA polymerase III subunit α- It has role in replicating the DNA
- DNA polymerase III subunit ε- It has 5′ to 3′ exonuclease activity (proofreading)
- DNA polymerase III subunit θ- It acts as a stabilizer which stabilizes the activity of ε subunit.
Other than these subunits of core enzyme, following are some other parts and their role in the DNA pol 3:
- 2 β units- It is the beta clamp which helps in joining the DNA pol 3 with DNA strand.
- 1 γ unit- This is the clamp loader which loads the clamp on the DNA strand.
DNA Polymerase 4 and 5
These two polymerases are involved in the DNA damage repair, i.e. SOS repair. As these strands do not have proofreading activity, therefore they produce mutated DNA even after a repair which is called as translation synthesis and adaptive mutagenesis.
Other than DNA Polymerases, there are some enzymes that are also involved in DNA replication of Prokaryotes which play an essential role.
Helicases are the enzymes that help in unwinding both the strands of DNA for the process of DNA replication to take place. As we know that DNA replication is semi-conservative, therefore, the 2 strands of the DNA which are held by hydrogen bonding needs to be separated before other enzymes like DNA polymerases can act upon on of the strand and make its copy.
Helicase enzyme helps in separating the strands by breaking the H-bonding between the bases of the two strands. There are 3 types of helicases in Prokaryotes, viz;
- DNA A– It only opens up the Ori site, that is the point where DNA replication begins.
- DNA B– This opens up the main DNA strand.
- DNA C– This acts as a loader of both DNA A and DNA B.
SSBP (single-stranded binding protein)
SSBPs are the enzymes that help in keeping the separated DNA strands separated. That is, they prevent the rewinding of the DNA strand which has been separated by helicase. These enzymes are like small beads that attract the nucleotides and prevent the formation of bonds.
They maintain the single-stranded structure of DNA and also protect DNA from nuclease activity (DNAse).
Primase is involved in the synthesis of Primer which is a short nucleotide sequence and acts as an initiator to the DNA replication. In the case of prokaryotes, DNA G act as Primase.
This enzyme helps in joining the two strands of the DNA. It is used for sealing the gaps between the fragments of DNA, for example, okazaki fragments. They require energy for this activity in the form of ATP and NAD+.
This enzyme removes the supercoiling of the DNA strand. They can either overwind or underwing the DNA strand. The problem of DNA supercoiling arises during replication with the action of helicase. Therefore supercoils are always generated ahead of the replication fork. Imagine opening 2 strands of ropes which are coiled together, this unwinding of the ropes will lead to a tension in the upper end, this tension formed is called as supercoiling and is removed by topoisomerase which introduces cuts in either one or both the strands of the DNA and then unwinds and join them. E.g Gyrase.