Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies. Kenneth N Kreuzer

      Figure 3.1. The Escherichia coli replicative DNA polymerase. The three-subunit core of the bacterial replicative polymerase (panel A, left) has active sites for polymerase and exonuclease activity (in separate subunits; the open mouths), and a structural subunit (the hat). The complete DNA polymerase complex, called holoenzyme, is diagrammed at the right of panel A and consists of two copies of the core polymerase along with clamp (panel B) and clamp loader (panel C). The sliding clamp is a ring-shaped protein consisting of two identical subunits (red and blue in panel B), each of which has three similar subdomains. The image of the crystal structure of the clamp shown at the left of panel B is from the RCSB PDB (www.rcsb.org) of PDB ID 2POL (Kong et al., 1992). The C-terminal face of the clamp, which binds both clamp loader and DNA polymerase core, is facing up in the cartoon diagram on the right of panel B. The clamp loader (panel C) consists of five subunits derived from three different genes, as described in the text. Two of the subunits, the ones with the extended arms, bind to the core of the DNA polymerase in the holoenzyme. The clamp-loading pathway is diagrammed in panel D. Parts of this figure were modified from Kelch et al. (2012).

      The subunits of the holoenzyme are named after letters of the Greek alphabet. Detailed discussions of the roles of every subunit are beyond the scope of this chapter, but for those who engage the literature further, the names of the subunits are as follows: polymerase III core is composed of α, ε, and θ; the clamp loader is composed of τ, γ, δ, and δ′, and is also called the γ complex; the clamp is called β, and the architectural proteins are χ and ψ.

      The three-subunit polymerase III core complex that replicates both the leading and lagging strand has separate subunits with DNA polymerase and proofreading exonuclease activity (unlike the combined activities within the major subunit of the bacteriophage T7 DNA polymerase). When misincorporation occurs during DNA synthesis by polymerase III core, the growing DNA chain with the errant nucleotide residue is therefore passed between the two protein subunits to excise the incorrect base and then back to the polymerase subunit to resume DNA synthesis. The third subunit of the polymerase core plays a structural role and assists the proofreading subunit, and in some bacterial species, this subunit is missing.

      3.3Dynamics of clamp loading in bacterial replication

      The sliding clamp was uncovered as a protein factor that greatly increases the processivity of replicative DNA polymerase. Early studies of sliding clamps led to a remarkable discovery. When clamps were loaded onto circular DNA, they remained bound to the DNA indefinitely, but if the DNA was cut with a restriction enzyme to form linear DNA, the clamps quickly fell off (see “How did they test that?” at the end of this chapter). This led to the model that clamps encircle the DNA like donuts with a string through the donut hole and can readily slide along the DNA. The eventual solution of sliding clamp structures confirmed this view, showing a beautiful donut-like structure with a hole large enough to accommodate duplex DNA (Figure 3.1B).

      A quick glance at the crystal structure of the E. coli sliding clamp reveals what appears to be a hexameric complex — six very similar structures make up the donut structure. However, there are actually only two subunits, each of which has three domains of similar structure. Several features of the clamp structure are notable. First, α helices line the central hole, providing strong electrostatic potential that allows water-mediated interactions with DNA. This arrangement allows free movement of the DNA through the hole in the clamp. Second, the C-termini of both subunits are exposed on one face of the clamp, and this is the site of interaction of the clamp with other proteins including the DNA polymerase and the clamp loader. Third, in spite of the similar structures of the three domains in each subunit, they have very different amino acid sequences. Finally, clamps from a bacteriophage called T4 and from eukaryotic cells have a quite similar structure with six domains in a donut shape, but in contrast, are formed from three subunits with two domains each (see Chapter 4).

      As already introduced, the major function of the sliding clamp is to increase the processivity of the replicative DNA polymerase. The processivity of replicative polymerase is quite a challenging problem in DNA replication, since the leading strand is synthesized continuously for as many as millions of base pairs, while lagging-strand synthesis must be stopped, within each Okazaki fragment, after only a thousand or so base pairs (in bacteria) or a few hundred base pairs (in eukaryotes). The dynamics of the sliding clamp are critical for the careful activation and termination of DNA polymerase action at appropriate places in the replication fork to achieve this distinct behavior on the leading and lagging strands.

      The clamp loader complex within the holoenzyme plays the key role of loading a new clamp for each Okazaki fragment (Figure 3.1C and 3.1D). During each round of Okazaki fragment synthesis, a clamp is loaded onto the short RNA primer made by primase (see below) and the clamp is then handed off to the replicative DNA polymerase. Being associated with clamp, the polymerase now has a high level of processivity so that it can synthesize the Okazaki fragment to completion.

      To accomplish its loading reaction, the clamp loader must bind to the clamp when it is free in solution, deliver the clamp to the DNA target, and then lose its affinity for the clamp so that the clamp can associate with the DNA polymerase. During this reaction sequence, the clamp loader must direct the donut-shaped clamp through a key transformation — opening up the donut to allow DNA to enter the central hole and then allowing the donut circle to reform as the loader dissociates from the clamp (Figure 3.1D).

      The structure of the clamp loader helps to explain the intricate gymnastics of this loading reaction. In both bacteria and eukaryotes, the clamp loader is a five-subunit complex, and each subunit has a related three-domain structure. The two N-terminal domains together have the structure characteristic of proteins of the AAA+ family of ATPases (named after the rather generic description, ATPases associated with various cellular activities), and the function of ATP binding and hydrolysis is to fuel the conformational changes needed to carry out the abovementioned steps in the loading reaction. Members of the AAA+ family typically adopt a hexameric ring structure, but the five clamp loader ATPase modules adopt a configuration like a hexamer missing one subunit. This reveals a C shape with a gap between two of the subunits and a hole near the bottom (Figure 3.1C and 3.1D). The remaining C-terminal domains of the clamp loader subunits form a tight pentameric cap at the top end of the clamp loader.

      Upon binding to ATP, the clamp loader binds the C-terminal face of a free clamp on the bottom of the loader — the binding reaction pries open one of the subunit–subunit interfaces of the clamp, cracking the donut (Figure 3.1D). This gap in the clamp is sufficiently large to allow the passage of DNA. The clamp–clamp loader complex has a high affinity for the site where RNA primer has been synthesized, specifically binding to the 3′ end where DNA synthesis must begin (this is called the primer-template site). This binding occurs in an interesting fashion — the interior of the clamp loader is organized to bind duplex DNA lengthwise, but the cap forces a tight bend as the DNA approaches. SsDNA is much more flexible than duplex DNA, and so a primer-template junction easily forms the bent structure required, positioning the clamp loader at the correct site (Figure 3.1D). Notice that as the clamp loader binds the primer-template junction, the duplex DNA has been threaded into both the interior of the clamp loader and simultaneously through the crack in the clamp. Also notice that the clamp is positioned in a specific orientation, with the side that binds clamp loader (the C-terminal face) pointing in the same direction as the 3′ end of the primer. Once bound to the primer-template