DNA dependent DNA polymerase

The genetic material DNA is synthesized by an enzyme called DNA dependent DNA polymerase that uses parental DNA strand as a template, on which new DNA will be synthesized. Both prokaryote and eukaryote cells contain a large number of DNA polymerases. Some of these are involved in replication, while some polymerases play a major role in repair and recombination process. Interestingly few enzymes catalyze the synthesis of a DNA strand using RNA as complementary strand are called as RNA-dependent DNA polymerase (reverse transcriptase). The existence of RNA-dependent DNA polymerases was first demonstrated by Temin and independently by Baltimore.

The DNA polymerases which are involved in vivo (within the cell) replication process are called replicases.

Example: DNA polymerase-III in E-coli, DNA polymerase γ (gamma) in mitochondria and DNA polymerase α, δ and ε in eukaryotes.

 

DNA polymerase (replicase) has the following features

  1. Replicases are relatively less abundant in the cell compared to other DNA polymerases
  2. These are distributive enzymes that can bind the template strand and capable of incorporating the several thousands of nucleotides in growing DNA molecules before they release from the template.
  3. They have relatively higher Vmax compared to other polymerases.
  4. However, mutations in these enzymes alter the V max and sometimes lethal and therefore the mutations can be studied only by creating conditional mutants.

 

I) Prokaryotic DNA polymerases

Arthur Kornberg, who was won the noble prize in 1959 for isolation of polymerase from E. coli in 1956. As this enzyme has the ability to incorporate deoxyribonucleotides, Kornberg named this enzyme as DNA polymerase which was later renamed as DNA polymerase-1 or Kornberg enzyme.

DeLucia and Cairns isolated the DNA polymerase-I mutant E. coli strain and surprisingly they found that the mutant strain synthesized DNA normally. This interesting discovery was created doubts on the role of DNA polymerase in replication and led groups to search for other replication enzymes. Finally, Cairns isolated the other types of DNA polymerases which are involved in DNA replication in DNA polymerase-I mutant E. coli strain such enzymes are DNA polymerase-II and DNA polymerase- III from the same strain.

At the same time, Klenow and his colleagues showed that the treatment of DNA polymerase with the proteolytic enzyme subtilisin significantly increased the polymerase activity and decreased the exonuclease activity. The resulting DNA polymerase was isolated and was named as “Klenow fragment”.

DNA polymerase -I, II and III of E. coli strain are remained in the active state under normal conditions, while DNA polymerase-IV and V remain in the inactive state until they are activated by SOS repair mechanism.

DNA polymerase -I is the most abundant enzyme about 400 copies per cell. The resulting enzyme involved mainly in DNA repair and also has the primer excision activity, that is used in removing of primers during replication.

 

DNA polymerase -I contains three activities in the biological system they are….

  1. 5′ to 3′ polymerase activity or gap filling activity
  2. 3′ to 5′ exonuclease activity or proof reading activity
  3. 5′ to 3′ exonuclease activity or primer excision activity (this activity unique for polymerase-II).

As mentioned above, when DNA polymerase-I treated with proteolytic enzyme trypsin or subtilisin, the DNA is separated into the large domain (Klenow fragment) and a smaller fragment contain 5′ to 3′ exonuclease activity.

The large domain such as Klenow fragment contains 5′ to 3′ polymerase activity, 3′ to 5′ exonuclease activity but lacks 5′ to 3′ exonuclease activity.

DNA polymerase-I mutants are highly sensitive to UV- radiation and other mutagens.

DNA polymerase-II is a dimeric enzyme and coded by pol-B gene, it is involved in DNA repair but any mutations in a pol-B gene will not have any significant effect on the bacterium because the activity of polymerase-II substituted by polymerase-I.

In E. coli DNA polymerase-III is the most complex enzyme compared to other DNA polymerase complexes.

 

There are at least ten different sub-units in the enzyme that can be divided into the following units.

1)  Core enzyme– α, ε and ϴ

α- 5′ to 3′ polymerase

ε- 3′ to 5′ exonuclease

ϴ- increases the affinity of the enzyme for DNA

2)  Sliding clamp- β2 Responsible for distributing residue of the enzyme

3) Clamp load complex- γ- complex- form sliding clamp contains 2 or 3 subunits of γ, one subunit each of δ, δ’, ѱ and χ ATPase complex.

4) Dimerizing subunit- Г dimerizing of the two polymerases involved in leading and lagging stand synthesis.

under normal condition DNA, polymerase-IV and V remain in the inactive state due to the suppression by Lex-A.

When SOS repair is induced, the recombination protein Rec-A promotes proteolytic activity that cleaves Lex-A and there by activating DNA polymerase-IV and V.

DNA polymerase-IV and V are involved in the transmission synthesis are error-prone replication in which the polymerase randomly incorporate the nucleotides against DNA template region having errors.

 

II) Eukaryotic DNA polymerases

The nucleus of a eukaryotic cell contains a large number of polymerases, of these polymerases’ poly-α, δ and ε are involved in invivo replication of nuclear DNA and susceptible to Aphidicolin.

Localization of different DNA polymerases and their function have been described in the table.

S. No DNA polymerase Localization Exonuclease activity Function
3′ to 5′ 5′ to 3′
1 α (alpha) Nucleoplasm No  

No

Involved in the synthesis of primer and leading strand
2 β (beta) Nucleoplasm No  

No

DNA repair and gap filling
3 γ (gamma) Mitochondria (Matrix) Yes  

No

Mitochondria replication
4 δ (delta) Nucleoplasm Yes  

No

Lagging strand synthesis
5 ε (epsilon) Nucleoplasm Yes No Leading strand synthesis
6 λ (lamda) Nucleoplasm Recombination of SOS
7 κ (kappa) Nucleoplasm Required for attachment of cohesin

None of the eukaryotic nuclear DNA polymerases contains 5′ to 3′ exonuclease activity, so primer excision during replication will be done by RNase H and FEN1 complex.

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