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 surrounds DNA and slides along it while the Pol III holoenzyme is attached 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.