Types of DNA polymerase in Prokaryotes

DNA polymerase synthesize complementary daughter strand by reading the DNA template.

Introduction:

Bacterial DNA replication is a vital event in which the cell makes a copy of its genetic material with the help of DNA polymerase. The newly replicated DNA is transferred to the next generation via cell division. The principle of DNA replication is a semi-conservative type, where it synthesizes two identical copies of double stranded DNA, each with one new and one old strand. The process of copying the genetic material is not as simple as it sounds. It involves many proteins, enzymes and cofactors. The coordinated functions of proteins and enzymes assure error free DNA replication.

The key enzyme in DNA replication is DNA polymerase. It reads the parent strand information and synthesizes complementary daughter strand. The complementary relationship is followed as per the Chargraff’s and Watson & Crick pairing rule.

Characteristics of DNA polymerase:

1. It is primer dependent.
2. It cannot initiate the polymerization by its own.
3. The mechanism of polymerization is by adding free nucleotides to the 3’OH end of the primer and elongates it.
4. The proof reading ability of DNA polymerase ensures error free copying.
5. The polymerase enzyme adds nucleotides and get dissociates. The average number of nucleotides that adds DNA polymerase before dissociating is called as processivity.

Discovery of DNA polymerase:

The first DNA polymerase was isolated and discovered by Arthur Kornberg and his team. He has contributed significantly in isolation and purification of enzymes and coenzymes. While working on different enzymes, Arthur was keen to study the enzyme that could synthesize nucleic acid.  Discovery of DNA structure showed that nucleotides are the repeating units or monomers of nucleic acid strands.

The discovery and studies of polynucleotide phosphorylase by Mariane Grunberg-Manago showed that the enzyme is capable to synthesize RNA in test tubes on the availability of free nucleotides and ADP as coenzyme. This research ignited the curiosity and for the enzyme that could synthesize DNA. With the help fermenter, Arthur obtained E.coli log phase culture and isolated the E.coli extract. By using chromatographic techniques, he isolated and purified DNA polymerase enzyme. The Enzyme that Arthur isolated is called as DNA polymerase I. Thomas Kornberg; son of Arthur Kornberg continued the legacy of enzyme isolation and purification. He also isolated two DNA polymerase from E. coli.

Types of DNA polymerases found in E. coli DNA polymerase I-

DNA Polymerase I

1. It was discovered firstly (1955) by Arthur Kornberg.
2. It was isolated from E. coli
3. It’s molecular weight is 103,000
4. It is encoded by pol A gene
5. It is single polypeptide chain
6. It works on phosphoryl group transfer. The 3’OH of primer nucleophilic attack the 5’ phosphate of upcoming nucleotide.
7. It elongates DNA from 5’ to 3’ direction.
8. It has 3’ to 5’ exonuclease activity also called as proof reading.
9. It has 5’ to 3’ exonuclease activity
10. The 5’ to 3’ exonuclease activity enables the enzyme to remove RNA primer from DNA and RNA hybrid. It is called as Nick translation.
11. It can add 3 to 200 nucleotides when it associates with DNA, hence it has low processivity.
12. The polymerization rate is 16 to 20 nucleotides per second; hence it is not the primary polymerase enzyme.
13. When DNA polymerase I is treated with protease, it forms two fragments. The one fragment is large and it retains the polymerization and proof reading function. This large fragment is called as Klenow fragment. The other small fragment retains the nick translation i.e. 5’ to 3’ exonuclease activity.

DNA polymerase II

1. it was discovered in 1959 by Thomas Kornberg.
2. It was isolated from E. coli
3. The molecular weight of the polymerizing subunit is 88000.
4. It is encoded by pol B gene.
5. It has 7 subunits (polypeptides chains).
6. It works on phosphoryl group transfer. The 3’OH of primer nucleophilic attack the 5’ phosphate of upcoming nucleotide.
7. It elongates DNA from 5’ to 3’ direction.
8. It has 3’ to 5’ exonuclease activity also called as proof reading.
9. It does not have 5’ to 3’ exonuclease activity.
10. The polymerization rate is 40 nucleotides per second.
11. The DNA polymerase II when once associate with DNA, it can add up to 1500 nucleotides.
12. The major role of DNA polymerase is in DNA repair.

DNA polymerase III –

1. It was discovered in 1970 by Thomas Kornberg
2. It was isolated from E. coli
3. The molecular weight of polymerizing subunit is 791500.
4. It is encoded by pol C gene.
5. It has 10 subunits (polypeptides chains).
6. It works on phosphoryl group transfer. The 3’OH of primer nucleophilic attack the 5’ phosphate of upcoming nucleotide.
7. It elongates DNA from 5’ to 3’ direction.
8. It has 3’ to 5’ exonuclease activity also called as proof reading.
9. It does not have 5’ to 3’ exonuclease activity
10. The polymerization rate is 250 to 1000 nucleotides per second.
11. The DNA polymerase II when once associate with DNA, it can add up to 5,00,000 nucleotides.
12. It is the primary DNA polymerase enzyme

Structure of DNA polymerase III –

It looks like right hand of humans, consisting of thumb, finger and palm domains. Different domains are involved in different functions. Thumb domain is responsible for processivity, translocation and positioning of DNA. Finger domain binds upcoming free nucleoside triphosphates with the complementary bases of template. The Palm domain catalyzes transfer of phosphoryl groups.

DNA polymerase III holoenzyme consist of 10 subunits namely α, ε, θ, τ, γ, δ, δ’, χ, ψ and β. The subunits α, ε, θ forms a core polymerase. The DNA polymerase enzyme consist of two-core polymerase and their dimer is formed by τ. The γ complex consisting of τ₂γδδ’ subunits acts like clamp loading complex. The association of core polymerase enzyme ensures high processivity.

The functions of the subunits are as follows-

α – The alpha subunit is component of core polymerase enzyme. It is encoded by pol C gene

ε – The epsilon subunit does proof reading. It is also a component of core polymerase. DnaQ encodes it.

θ – Theta is part of core polymerase and is involved in assembling α and ε. DnaE gene encodes it

τ – Tau assist in binding to DNA template. It also plays a vital role in dimerization of Core polymerase enzyme. DnaX gene encodes it.

γ  – Gamma functions as a Clamp loader and it is encoded by DnaX gene

δ – Delta opens the clamp and it is encoded by holA

δ’ – Delta prime also acts as a clamp loader, it is encoded by holB

χ – Chi interacts with single stranded binding proteins. holC gene encodes it.

ψ – Psi interacts and coordinates with Gamma and Chi. It is encoded holD gene

 β – It is Clamp required for maximum processivity. It is encoded by holN gene

DNA polymerase IV & V –

1. It can polymerize DNA from 3’ to 5’ direction
2. It has no 5’ to 3’ exonuclease activity hence it lacks proof reading.
3. It is involved in DNA repair
4. Their genes are activated by SOS response.
5. The major function of DNA polymerase IV is to halt DNA polymerase III at the replication fork and provides time for DNA repair.
6. It is also found to be involved in DNA translesion synthesis, where the unrepaired or mutated parental sequence is copied and daughter strand is synthesized.

References:

https://www.whatisbiotechnology.org/index.php/science/summary/

Microbiology by Prescott, 5th Edition

Principle of Biochemistry by Nelson and Cox, 5^th edition

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC150442/

https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/replication/a/molecular-mechanism-of-dna-replicatioI