Читать книгу Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies - Kenneth N Kreuzer - Страница 32

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 junction, the ATPase activity of the clamp loader is activated, and the ATP-free clamp loader loses its affinity for the primer-template site. As the clamp loader dissociates from the primer-template and clamp, the clamp springs back to its donut shape, which now encircles the DNA. DNA polymerase also binds to the C-terminal face of the clamp, and so the clamp is oriented correctly on the primer-template junction to bind the replicative polymerase, which can now proceed with Okazaki fragment synthesis.

As mentioned earlier, clamp loaders from both prokaryotes and eukaryotes consist of five subunits, each being an ATPase family member. The subunit composition of the bacterial clamp loader is interesting. Due to redundancy, only three genes encode the five subunits of the E. coli clamp loader. Two of these genes encode a single subunit each, while the remaining three subunits are all derived from a single gene called dnaX. Recall from Chapter 2 that one particular T7 gene encodes two different forms of the helicase protein in bacteriophage T7. In a related way, the E. coli dnaX gene encodes two distinct proteins, but by means of a different mechanism. In this case, ribosomes translating the dnaX mRNA sometimes engage in a process called programmed translational frameshifting, in which the reading frame is suddenly shifted to change the register of the triplet code downstream of the frameshift event. Shortly after the frameshift occurs, the ribosome encounters a stop codon, resulting in a short (47 kDa) form of the protein. Most of the translation events do not result in a frameshifting event, and these lead to the long (71 kDa) form of the protein. Both forms of the protein are competent for binding the clamp and the other subunits of the clamp-loader complex, but only the long form has the domain required for binding DNA polymerase and the replicative helicase. Recent biochemical experiments argue that the normal replisome contains two of the long forms and one short form, accounting for the three subunits encoded by dnaX. This stoichiometry is important, because the two long forms of the protein are able to contact two polymerase core complexes, one for the leading strand and one for the lagging strand. Note that the clamp loader thereby plays the key role of tethering and coordinating the two DNA polymerase core complexes within the replisome.