How does the structure of E. coli DNA Polymerase III relate to its function?

AUTUMN MB II Essay Philana Fernandes

DNA polymerase III is an asymmetrical dimer (that is, it lacks inherent symmetry) one half of which is devoted to synthesizing the leading strand at the advancing replication fork and the other, the lagging strand. It is composed of 18 subunits in total (only ten of which we shall go on to discuss as their functions are understood), from only ten structural genes. Thus, more than one subunit is produced from one gene – for example, subunit tau and gamma are produced from the same gene, indeed they are both motor ATPases.

The sequence of events is as follows

The β dimer and the clamp loading complex bind to template DNA to form what is known as a pre-initiation complex

This pre-initiation complex then binds with high affinity to DNA polymerase III core enzyme (α, θ and ε), which so facilitates the binding of a second core polymerase enzyme to form the holoenzyme.

There are three sub-assemblies within DNA polymerase III – the catalytic core (three subunits, α, θ and ε); the sliding clamp (the β subunit) and the clamp loader complex (the delta, the delta prime, the gamma, the chi and the psi subunit.) In addition, the tau subunit serves as a scaffold to help in the dimerizaton of the core complex through the two α chains of the separate core enzymes by sequence homology with the gamma subunit.

The core enzyme has very low processivity - only 10-15 residues. Thus, it is only able to ‘fill-in’ short regions of single stranded DNA. The holoenzyme is created from seven other subunits (β, delta, delta prime, chi, gamma, psi, and tau) that bind to the core. The holoenzyme has the full functions of DNA polymerase III – polymerase, proofreading and processivity activities.

It is necessary for two subunits conferring polymerase activity to be in a single complex as this ensures that both leading and lagging strands are synthesized at the same rate. Both halves of the dimer contain in the core, an α subunit (the subunit providing the polymerase activity of1160 residues) and a ε subunit which gives the holoenzyme the 3’-5’ proofreading exonuclease activity. Proofreading aids the maintenance of high replication fidelity i.e. by reducing the number of mis-encorporated nucleotides. The core also contains a theta subunit: its function is not yet fully understood although presently studies using nuclear magnetic ...

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Recently, NMR spectroscopy has lead to the determination of the ε subunit structure. It was found to have three a-helices in the N-terminal two thirds of the protein that fold to form a triangular shape - this is the catalytic domain. The similarity of this type of structure to the ring-shaped clamp of the beta processivity clamp is remarkable and indicates that the epsilon subunit also ‘reads the DNA’ by letting it flow through the triangle of its N terminus. The C-terminal section of theta has many charged and hydrophilic amino acid residues with no well-defined secondary structure and exists in a highly dynamic state, this is the α binding domain.

The β subunits clamp the core polymerase to the DNA enabling processivity of the holoenzyme. The subunit is a ring-shaped clamp that embraces DNA in a central 35-angstrom hole and tethers the core polymerase III to the template. The 35 angstrom hole of the β dimer is not only large enough to accommodate the whole of the B type double helical DNA due to minimised steric hindrance but is also lined with positively charged residues such as lysine and arginine so allowing for an attraction between the channel and the DNA phosphate backbone.

In particular, there are three arginine residues that are of particular importance – Asp 256 aids to stabilise the transition between the monomer and the dimerizaton state, whilst Asp 190 and 192 help to position the incoming nucleotide.

There are 12 central α helices within the β subunit that are tilted – this means that the axis of each of these α helices are at right angles to the sugar phosphate backbone of the DNA helix – both in its major and minor grooves. This is in direct contrast to other known DNA binding proteins that contain α helices. In these, the helices are usually found to lie parallel to the nucleic acid backbone. Thus, it seems that the perpendicular orientation of the beta clamp helices and DNA backbone are designed to make access of the protein to either DNA groove difficult. Thus, rapid sliding of the clamp along the DNA axis is instead facilitated. In support of this theory is the fact that the interaction between β subunit with the DNA-RNA duplexes- found at the site where initial clamping of the β subunit occurs at the RNA-primed template at the start of the Okasaki fragment.

Since β is a closed protein ring, a clamp loader is required to open the ring and load it onto DNA for use by the core enzyme. In E. coli, the multi-subunit gamma or clamp loading complex, catalyses β dimer assembly on DNA in a reaction that requires ATP

Of the three different subunits of the gamma complex, delta is the only one that is capable of binding to and opening the beta clamp on its own. This gives support to the theory that the delta subunit acts as a ‘wrench’ in either inducing or maintaining a conformational change in the beta monomers at the dimer interface so that ring closure becomes unfavourable. In addition to this, delta reduces the curvature of each of the monomers in the open state relative to the monomers in the dimer.

However, it is also believed that beta is not completely passive in controlling its open state – it is thought to have a ‘spring-loaded’ tendency to adopt the open conformation so supplementing the action of the delta wrench.

The clamp-loading complex utilises ATP hydrolysis in order to provide the necessary energy for the conformational changes required in the delta subunit, these are provided by gamma subunit, a ‘motor’ ATPase.

Binding of ATP also ensures high affinity of the clamp-loading complex for the primer-template DNA, bringing the open clamp and DNA in close proximity to each other. As the β clamp then goes on to bind with high affinity to the core enzyme, the increased affinity of the clamp loading complex for the primer template DNA helps not only in the loading of the core polymerase but also in the unloading of the core polymerase from the β dimers on the lagging strand. When an Okasaki fragment is completed, the used clamp loading complexes lack ATP and so the affinity for the DNA-primer template and thus are free to dissociate from the DNA, in order to associate with it again at a point upstream.

The gamma complex then hydrolyses the ATP molecules one at a time- these sequential ATPase reactions act to place DNA within the open clamp, close the clamp around DNA and, finally the release of the topologically linked clamp and DNA.

Thus, the 10 subunits discussed use a variety of structural elements to aid function. Sequence homology favours dimerizaton (seen clearly in the β clamp but also in tau and gamma subunits); the polymerase as a whole is essentially repeated twice (with the exception of the dimerizaton subunit, tau) to account for the two strands which must be synthesised separately, and the beta clamps inherent structure especially its α helices are positioned to aid the movement of the DNA through it (this has been visualised with electron microscopy). It is thought that the other subunits which are not yet fully understood will reveal more structures aiding the E.coli DNA polymerase III activity.

Together they enable E. coli DNA polymerase III to achieve a rate of DNA replication of 1000 nucleotides per second with only a 1/10 10 error rate.