Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies
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.
3.4Coordinated action of helicase, primase, and DNA polymerase holoenzyme
As in the case of bacteriophage T7, the proteins in the bacterial replisome are carefully coordinated and function together as a protein machine (Figure 3.2A). The DNA polymerase holoenzyme described above interacts with the replicative helicase, directly linking the unwinding of the parental DNA strand with the synthesis of the leading strand. Thus, both clamp and the replicative helicase assist the leading-strand DNA polymerase in its rapid and processive synthesis reaction around the bacterial chromosome. The structure of an intact functioning E. coli replisome has yet to be deduced, and the number of interacting subunits is greater than that in the phage T7 system. For these reasons, our understanding of the E. coli replisome is not quite to the level of that of the T7 replisome.
The E. coli replicative helicase has many similarities to that of bacteriophage T7. It is a hexameric protein that unwinds DNA using the energy of nucleotide hydrolysis, and it tracks along ssDNA in the 5′ to 3′ direction, placing it on the lagging-strand template (Figures 3.2A and 3.3A). It has a ring or donut-like shape, and the lagging-strand template passes through the hole as the leading-strand template is unwound and passes around the outside of the helicase. As in the T7 system, unwinding activity of E. coli replicative helicase is stimulated when it is associated with the DNA polymerase complex. This reveals a general feature in which replicative polymerases and helicases reciprocally stimulate activities of each other within the replication complex.
Figure 3.2. The Escherichia coli replication machinery. The DNA polymerase holoenzyme complex links the leading and lagging strands, with one copy of the core polymerase on each (panel A). The clamp-loading complex within the holoenzyme repeatedly loads sliding clamps (gray rings) as replication progresses. The helicase (orange) and primase (light blue) form a complex at the front of the replication machinery, unwinding the parental duplex. ssDNA-binding protein (green) covers all ssDNA on the lagging strand. On some occasions, blockage of the leading-strand polymerase can lead to re-priming of synthesis on the leading strand (panel B), rescuing an otherwise stalled replication fork.
While the E. coli replicative helicase does not itself possess primase activity, it does associate with the E. coli primase enzyme, again placing the primase in the proper location to synthesize RNA primers for Okazaki fragment synthesis on the lagging strand (Figure 3.2A). The functional unit for binding primase consists of two helicase subunits, and so the hexameric helicase can bind no more than three primase monomers at a time. The bacterial primase, like that of T7, has a weak sequence selectivity, with a preference for initiating primers using the sequence 5′-AG. The bacterial enzyme differs from that of T7 in synthesizing longer RNA primers, roughly 10–12 nucleotides long.
The bacterial helicase has some additional distinctive features compared to the T7 helicase. The E. coli enzyme, like most other DNA helicases, hydrolyzes ATP, whereas you will recall that the T7 enzyme hydrolyzes dTTP. In addition, the hole in the middle of the bacterial enzyme has a diameter that is larger than the diameter of a DNA double helix, and in fact, duplex DNA can slide through the central hole if it is appropriately loaded onto the enzyme. This accounts for an interesting activity of the protein, namely its ability to knock some duplex DNA-binding proteins off DNA (Figure 3.3B). Obviously, the helicase must be loaded appropriately onto DNA in order to catalyze the DNA unwinding at the replication fork and pass only a single strand of DNA through the donut hole. This brings us to another major difference from the T7 helicase. The E. coli helicase requires a special protein to get loaded onto DNA for unwinding (Figure 3.3C). When not bound to DNA, these two proteins form a tight complex, which is competent for bringing the replicative helicase to the special origin site where DNA replication initiates (see Chapter 5). This loading process is carefully regulated to prevent inappropriate helicase loading at random sites in the genome, which could cause partial replication of only portions of the genome.2
Figure 3.3.Functioning of the Escherichia coli replicative helicase. The hexameric replicative helicase unwinds DNA at the replication fork by traveling along the lagging-strand template in the 5′ to 3′ direction (panel A). The central channel in the helicase is large enough to accommodate duplex DNA, and the helicase can track along duplex DNA and displace bound proteins (panel B). Loading of the replicative helicase is carefully controlled by its association with a loading protein, which blocks the central channel when in solution but which helps deliver the helicase to appropriate structures such as forked DNA (panel C).
An interesting aspect of replication fork dynamics is that the helicase and primase need to travel in opposite directions along the lagging-strand template, even though they are bound to each other (E. coli) or are one and the same protein (T7). Recall that the helicase is moving along the lagging-strand template in the 5′ to 3′ direction, while the primase synthesizes the new strand in the 5′ to 3′ direction (which means it is traveling 3′ to 5′ on the lagging-strand template strand). This again requires careful coordination, in that the unwinding catalyzed by helicase needs to pause, while primase synthesizes the short RNA primer. Once the primer is synthesized, the clamp component of holoenzyme is targeted to the RNA primer site as described earlier. This is a key step in the handoff of the new primer to DNA polymerase for synthesis of the next Okazaki fragment.
During E. coli DNA replication, the lagging strand is looped around and the fork behaves according to the trombone model described in the previous chapter. As mentioned