DNA dependent DNA polymerase (DNAP of prokaryotes)
DNA polymerases are the enzymes involved in DNA synthesis from deoxyribonucleotides.
DNA polymerase adds nucleotides to the 3′- end of a DNA strand, one nucleotide at a time.
Chemical reaction catalysed:
What do you mean by processivity of DNA polymerase enzyme?
- The processivity of a DNA polymerase is the number of nucleotides added by the polymerase each time it binds to the template.
- The processivity of DNA polymerases vary with the type of polymerase and can range from few nucleotides to as many as 50,000.
- The processivity of DNA polymerases is increased dramatically by their association with the sliding clamps.
Structure of DNA polymerase from E. coli
The shape of the DNA polymerase resembles a right hand with thumb, finger, and palm domains.
1) The palm domain: The active site of polymerase resides in the palm domain. DNA is bound to the palm when the enzyme is active. Composed of β-sheet and binds two divalent metal ions like Mg2+ or Zn2+
Function: Catalyzing the transfer of phosphoryl groups in the phosphoryl transfer reaction.
2) The finger domain: The nucleotide recognition and binding with the template base.
3) The thumb domain: Plays a potential role in the processivity, translocation, and positioning of the DNA. Important role in binding of substrate DNA with polymerase enzyme.
Types of DNA polymerases in prokaryotes
Prokaryotes consist of five DNA polymerases of which only Pol III is involved in DNA replication.
The first polymerase to be identified was Pol I from E. coli. The enzyme was isolated by Arthur Kornberg.
DNA polymerase I
- Encoded by the polA structural gene. Molecular weight is 103,000 Dalton
- Pol I is the most abundant polymerase.
Polymerization rate: Pol I adds ~15-20 nucleotides per second.
Processivity: poor processivity (3 -200 nucleotides are added before it dissociates from the DNA template)
- 3’ to 5’ Exonuclease activity
- 5’ to 3’ Exonuclease activity
- 5’ to 3’ Polymerase activity
- 5’ to 3’ exonuclease removes the RNA primers at the 5’ ends of newly synthesized DNA while its DNA polymerase activity fills in the resulting gaps.
- The 5’ to 3’ exonuclease activity associated with DNA polymerase I degrade both single and double stranded DNA in the 5’ to 3’ direction, yielding 5’-mononucleotides. The 5’ to 3’ exonuclease activity is specific for double stranded DNA, yielding 5’-mononucleotides and oligonucleotides. DNA polymerase I can also excise mismatched regions in DNA.
Proof reading: If DNA polymerase III makes a mistake during DNA synthesis, the resulting unpaired base at 3′ end at the growing strand removed by the DNA polymerase I before synthesis continues.
- Polymerase I starts adding nucleotides at the RNA primer: template junction known as the origin of replication (ori). Approximately 400 bp downstream from the origin, the Pol III holoenzyme is assembled and takes over replication at a highly processive speed and nature.
- Fill gaps in DNA that arise during DNA replication, repair, and recombination.
Proteolytic cleavage of DNA polymerase I
- Polymerase I contains three separate active sites on a single polypeptide chain.
- The 5’ to 3’ exonuclease activity of Pol I is independent of its 3’ to 5’ exonuclease and its polymerase activities.
- The 5’ to 3’ exonuclease domain of the pol I can be removed by mild proteolytic treatment.
Protease used: Trypsin or subtilisin (cleavage site: peptide bond between 323rd and 324 th aminoacid residues)
Cleavage products: Smaller fragment (35KD) + Larger fragment (68 KD)
- The larger C terminal fragment is known as Klenow fragment.
- Klenow fragment retains its 3’ to 5’ exonuclease activity and 5’ to 3’ polymerase activity. But it lacks 5’ to 3’ exonuclease activity.
- Aminoacid residues in Klenow fragment: 324th aminoacid to 928th aminoacid
Smaller N terminal fragment has only 5’ to 3’ exonuclease activity
Aminoacid residues in smaller fragment: 1st aminoacid to 323 rd aminoacid
DNA polymerase II
- Encoded by structural gene polB.
- Molecular weight: 88000 Dalton
- Polymerization rate: 40 nucleotides/ sec
- Processivity: ~ 1,500 nucleotides are added before it dissociate from template
- 3’ to 5’ exonuclease activity: Proof reading function
- 5’ to 3’ polymerase activity
- Capable of synthesizing DNA on a damaged template (replication restart in UV-irradiated coli)
- Temporary function in DNA synthesis when polI and pol II are not functional.
- Participate in DNA repair (repair of DNA damaged by UV irradiation)
DNA polymerase III
- Encoded by structural gene polC (dnaE)
- Polymerization rate: 250 – 1000 nucleotides/ s
- Processivity: ~ 500,000 nucleotides are added before it dissociates from template.
- 3’ to 5’ exonuclease activity: Proof reading function
- 5’ to 3’ polymerase activity : Responsible for elongation process
DNA polymerase III holoenzyme
- DNA polymerase III holoenzyme is the primary enzyme involved in DNA replication in E. coli and belongs to family C polymerases.
- DNA polymerase III synthesizes nucleotide strands by adding new nucleotides to the 3’ end of growing DNA molecules.
- DNA polymerase III holoenzyme (Pol III holoenzyme) is the multi subunit replicase of the Escherichia coli chromosome. It contains 10 different subunits which assort into three functional components:
1) Core catalytic unit containing DNA polymerase activity
2) The β sliding clamp
3) Multi subunit clamp loader apparatus called γ complex (uses ATP to assemble the β clamp onto DNA).
1) Core catalytic unit:
Core polymerase can polymerize DNA but with limited processivity.
Composition: Consists of 3 subunits (α subunit, ε subunit and θ subunit)
1) α subunit function: It is the DNA polymerase (5’ to 3’ polymerase activity)
It has template binding and primer binding sites. It also contains an active site for free nucleotide binding next to the primer 3’end. The same site acts as a catalytic site for polymerization of properly base paired nucleotides from 5’ to 3’ direction.
2) ε subunit function: 3’ to 5’ exonuclease activity
It performs the proof reading function
3) θ subunit function: Act as a stabilizer for ɛ subunit.
The holoenzyme contains two cores (one for each strand – the lagging and leading strand).
2) Beta sliding clamp
- It is a ring‐shaped subunit that encircles DNA and slides along it while tethering the Pol III holoenzyme to the template.
- The β sliding clamp prevents the dissociation of DNA polymerase III from DNA.
- The beta sliding clamp processivity factor is also present in duplicate (one for each core). The β subunits associate in pairs to form donut-shaped structures.
3) Clamp loader complex (ϒ complex)
- The clamp loader is known as γ-complex in E. coli and has seven subunits.
- Sliding clamp loaders are protein complexes that are required for loading as well as unloading the sliding clamps on/from the DNA using energy from ATP hydrolysis.
Composition of ϒ complex: τ2γδδ′χψ
- Each holoenzyme consists of two copies of DNA Pol III core enzyme, two copies of beta sliding clamp and a copy of clamp loader (γ-complex).
- Each DNA Pol III core enzyme interacts with the γ-complex through τ protein. The τ protein consists of a flexible linker which allows DNA Pol III core enzymes to move independently.
Core polymerase + ϒ complex (without β subunit) = DNA Pol III*
- DNA polymerase III* can polymerize DNA, but with a much lower processivity.
- Two β dimers associate with the two cores within Pol III* to form the holoenzyme capable of replicating both strands of duplex DNA simultaneously.
Sliding clamp loading by clamp loading complex
- The ring‐shaped β clamp cannot assemble around DNA by itself.
- For this clamp loader is required which couples the energy of ATP hydrolysis to the assembly of the β clamp onto DNA.
- Using the energy from ATP hydrolysis, sliding clamp loading proteins open up the ring structure of the sliding DNA clamps allowing binding to and release from the DNA strand.
Once the complementary strand has been synthesized, there is decrease in the affinity of sliding clamp with the polymerase. This results in the release of the polymerase from the DNA and the sliding clamp.
The release of polymerase is not immediately followed by the release of sliding clamp. Rather various other proteins and factors become associated with the sliding clamp after the release of the polymerase.
These include the proteins involved in repair of okazaki fragments and chromatin assembly.
Steps involved in clamp loading by clamp loading complex:
- The clamp loader binds a molecule of ATP.
- Once the clamp loader has ATP molecule bound to it, it binds with the sliding clamp which results in the opening of the ring of the clamp.
- The opened ring sliding clamp is loaded onto the DNA: primer junction
- This binding results in the hydrolysis of ATP to ADP.
- This results in dissociation of clamp loader from the sliding clamp, and simultaneous binding of DNA polymerase.
- The sliding clamps are removed only when all the proteins and factors that act on newly synthesized DNA have interacted with the sliding clamp.
Common features of all class of prokaryotic DNA polymerases
- Synthesize any sequence specified by the template strand.
- Synthesize new strand in the 5’ to 3’ direction by adding nucleotides to a 3’-OH group.
- Use dNTPs to synthesize new DNA.
- Require a primer to initiate synthesis.
- Catalyze the formation of a phosphodiester bond by joining the 5’ phosphate group of the incoming nucleotide to the 3’-OH group of the preceding nucleotide on the growing strand, cleaving off two phosphates in the process.
- Produce newly synthesized strands that are complementary and antiparallel to the template strands.
- They are associated with a number of other proteins.
Proof reading activity of DNA polymerase
- DNA polymerase has an error-correcting activity called proofreading.
- DNA polymerase is so accurate that it makes only about one error in every 10^7 nucleotide pairs it copies.
- If an incorrect nucleotide is added to a growing strand, the DNA polymerase will cleave it from the strand and replace it with the correct nucleotide before continuing.
- Before the enzyme adds a nucleotide to a growing DNA chain, it checks whether the previous nucleotide added is correctly base-paired to the template strand. If so, the polymerase adds the next nucleotide.
- If base pairing is not correct, the polymerase removes the mis paired nucleotide by cutting the phosphodiester bond it has just made, releases the nucleotide, and tries again.
- Thus, DNA polymerase possesses both a 5’ to 3’ polymerization activity and a 3’ to 5’ exonuclease (nucleic acid–degrading) activity.
- These activities are carried out by different domains within the polymerase molecule.