DNA Polymerase III
DNA replication requires highly processive polymerases
- Replicative polymerases are characterised by their very high catalytic potency, fidelity and processivity.
- Processivity refers to the ability of an enzyme to catalyse many consecutive reactions without releasing its substrate.
- These polymerases are assemblies of many subunits that have evolved to grasp their templates and not let go until many nucleotides have been added.
- The source of processivity was revealed by determination of DNA polymerase III.
- This unit keeps polymerase associated with DNA double helix.
- It has star-shaped ring. A 32 diameter hole in its centre, which can readily accommodate a duplex DNA molecule and also leave enough space between DNA and the protein to allow rapid sliding during replication.
DNA polymerase III (E.coli main DNA replicase!)
- DNA polymerase I mutants can still replicate DNA
- DNA polymerase II discovered → DNA polymerase II mutants still function normally
- Mutant E.coli lacking DNA polymerase III → is lethal, therefore main replicase enzyme for E.coli!
- It is a multi-subunit complex (α, ε, θ, γ, Ψ, δ, δ', x, β) known as DNA polymerase III holoenzyme.
- Lacks 5' to 3' exonuclease activity (editing activity)
- There are 2 forms of the enzyme
- Core enzyme
- Holoenzyme
Core Enzyme
- Definition: Consists of only those subunits that are required for basic enzymatic activity.
- The DNA polymerase III core enzyme contains one each of the alpha, epsilon and theta subunits and can carry out the basic polymerase and exonuclease activities of polymerase III
- Consists of 3 subunits:
- alpha (α) → 5' to 3' (DNA Polymerase activity)
- epsilon (ε) → 3' to 5' (exonuclease proofreading activity)
- theta (θ)
- Ploymerisation and proofreading activities reside in its alpha and epsilon subunits.
- The theta subunit associates with a and E to form a core polymerase, which can polymerise DNA but with limited processivity.
- 2 core polymerases can be linked by another set of subunits, a clamp-loading complex, or y complex, consisting of 5 subunits of 4 different types (T2ySS') (also shown in pic below)
- The core polymerases are linked through tau (T) subunits.
- Two additional subunits x (chi) and Ψ (psi) are bound to the clamp-loading complex.
- The entire assembly of 13 protein subunits (9 diff types) is called DNA polymerase III.
DNA Polymerase III holoenzyme
- Multisubunit complex
- Consists of 17 polypeptides (make up 4 subassemblies)
- Core polymerase → 3 subunits: polymerase (a); 3' to 5' exonuclease (E); stimulator of the 3' to 5' exonuckease (θ)
- Subunit (T) → responsible for dimerisation (a compound formed by combination of two identical simpler molecules) of the core DNA polymerase.
- Sliding clamp → 2 homodimers (B), ring structure for processitivity
- 5 subunits have clamp-loader functions (δ‘,δ, γ, Ψ, x)
B subunits needed to increase processivity...
- DNA Polymerase III can polymerise DNA, but with much lower processivity than one would expect for the organised replication of an entire chromosome.
- This is because polymerase III core enzyme dissociates from template DNA after around 12 residues.
- Addition of B subunits (4 of which complete the DNA Polymerase III holoenzyme) → This is what increases processivity!
- Donut-shaped structure formed (as B subunits associate in pairs), this structure encircles the DNA and acts like clamps (as shown in the pic above)
- Each dimer associates with a core subassembly of polymerase III (one dimeric clamp per core subassembly) and slides along the DNA as replication proceeds.
- The B sliding clamp prevents the dissociation of DNA polymerase III from DNA, dramatically increasing processivity - to greater than 500,000!
- Therefore sliding clamp (B ring structures) increases polymerase III replication efficiency.
- Holoenzyme is able to add more than 5000 residues, and the B clamp keeps enzyme attached to the template.
- Y complex involved in loading B clamps onto template.
DNA polymerases of E.coli (slide27- might need to add more info!)
- Polymerase I and II and V function primarily in DNA repair
- Polymerase III - chief DNA - replicating enzyme of E.coli!