Seeing a molecular machine self-renew

Molecular machines are biological macromolecules or their complexes capable of generating nanometer-scaled physical movements required for cellular tasks. A wide variety of these nanomachines are found in nature, including those functioning in genome duplication and protein synthesis, named the replisome and the ribosome, respectively. In many ways, these biological nanomachines work just like a machine we use in our daily lives. They use the energy stored in molecular fuels to perform biologically useful work, for example, making new genetic materials as is the case for the replisome. Like a sophisticated clock that contains many moving parts, biological machines are composed of many minuscule moving components as well. The organization, coordination, and synchronization of these components govern the proper operation and the mechanism of how these components function together has fascinated many scientists. As illuminated by a study in PNAS (1), the mechanism of replisome self-renewal when a key component wears out (Fig. 1) is brought under the microscope using a duo of powerful, single-molecule manipulation and imaging techniques.

Fig. 1.

Mechanism of replisome self-renewal in T7 bacteriophage.

The report by Loparo et al. (1) focuses on one aspect of the dynamics in a working replisome from T7 bacteriophage, concerning its component exchange and processivity maintenance. Although this molecular machine has long been viewed as a stable, multiprotein complex that duplicates genomic DNA with a fast speed and proceeds a long way on the DNA before falling off or, in other words, with a high processivity, a more vibrant picture regarding the processes inside this black box has emerged in recent years (2). A structure much more dynamic than previously thought has been suggested for the replisome, with the possibilities of components exchanging, binding partners associating and dissociating, and even the entire complex reassembling after collapsing. The new study by Loparo et al. (1) illustrates an interesting mechanism that the replisome employs to ensure its high processivity. By recruiting excess polymerase molecules as backups and making replenishments when necessary, the replisome can keep its engine running even when the leading-strand polymerase falls off or, in simple terms, wears out.

Studying the composition of a complex molecular machine when it is running requires the capacity for multidimensional, simultaneous observations and the replisome is no exception. To examine the polymerase composition in an actively synthesizing replisome, Loparo et al. strategically combine two versatile single-molecule manipulation and visualization methods (1). First, a laminar flow of buffer is applied to stretch surface-tethered DNA molecules. As the replisome synthesizes along the leading-strand template, the DNA duplex remaining ahead of the replisome is shortened. This progression of the running replisome is followed in real time by tracking the location of a quantum dot (QD) placed on the duplex. Second, an organic fluorophore is attached to the polymerase. As it arrives at or departs from an active replisome, a characteristic fluorescence signal appears or disappears, respectively. The polymerase composition and identity are thus observed right at a synthesizing replisome. By combining these two approaches together, one can keep track of the function and composition of a replisome in situ at the same time.

Does a working replisome contain only one copy of polymerase for the leading strand? Using their combinatorial approach, Loparo et al. observe that besides the polymerase originally in place for the leading strand, additional copies of the polymerase can be associated to the replisome (1). These seemingly superfluous components then remain on an actively running replisome for tens of seconds.

Are these excess components simply spectators on a working replisome? To examine whether these newcomers can participate in DNA synthesis by the replisome, Loparo et al. use a catalytically slower mutant of the polymerase in a mixture with the wild-type polymerase, so that a polymerase exchange event can be detected as a sudden change in synthesis rate. Examining the time trajectories of the running replisome when both polymerases are supplied reveals the presence of synthesis by both types of molecules, one at a time, within individual time records. This observation suggests that the polymerase originally in place can be replaced by a newcomer that can participate in replication, leading to an acceleration or a deceleration in DNA synthesis rate. This exchange process does not occur instantaneously after the newly arrived component is associated to a running replisome. A waiting time of ∼55 s (1) is observed for the wild-type polymerase reaching a running replisome equipped with the mutant polymerase originally. However, it is also observed that for some molecules, the working replisome does not use the excess components on standby, but rather lets them dissociate after ∼44 s. Nevertheless, the excess components are not always idling, but can replenish the leading-strand polymerase when the latter falls off. Loparo et al. suggest that even an apparently fruitful polymerase exchange event does not result from an active replacement of the leading-strand polymerase in the replisome engine by the newly arrived polymerase backup, but rather reflects a self-disengagement step of the old polymerase followed by some kind of replenishment by the new molecule.

These exchange processes ensure that when the key component of a running replisome, the leading-strand polymerase, wears out, another similar component located nearby can kick in efficiently to keep this molecular machine running without undesired stalls. This process is observed for 75% of the replisome molecules when a pool of free polymerase is present with a 20-nM concentration. Which component of the replisome is the unsung hero that mediates this self-renewal process? Loparo et al. find that using a C-terminal truncation mutant of T7 helicase instead of the wild-type protein decreases the replisome processivity from 15.2 to 5 kb, whereas challenging the replisome containing wild-type polymerase and helicase with an excess of the polymerase mutant mentioned above gives a similar decrease in processivity to 3.3 kb. This similarity in processivity suppression is connected to a possible role of the truncated region of the helicase in facilitating polymerase exchange. In a previous work by Hamdan and coworkers (3), the C-terminal tail of the helicase has already been found to interact with the polymerase. Moreover, the current study shows that truncating this region at the C terminus from the helicase leads to a sharp decrease in the percentage of replisome exhibiting the polymerase exchange (1), underscoring the critical role of this region of the helicase in the self-renewal of the replisome.

Using the clues obtained from the current study, Loparo et al. piece together a unified dynamic picture of the replisome composition, processivity, and self-maintenance (1). On one hand, the C-terminal region of the helicase serves as a safety net in preventing an actively synthesizing replisome from losing its key component, the leading-strand polymerase, by direct contact. On the other hand, this same region, with vacancy present given the six binding sites available on the hexameric helicase,

Loparo et al. piece together a unified dynamic picture of the replisome composition, processivity, and self-maintenance.

recruits extra copies of the polymerase to the replisome and keeps them on standby for sufficiently long, so that when the running replisome loses a polymerase, a backup copy can quickly fill the vacancy. These mechanisms nicely reconcile the apparently incompatible evidence from previous ensemble biochemical experiments that concern the high polymerase processivity observed even with dilution (4–7) and the polymerase exchange detected when excess polymerases are in solution (8, 9).

This work clearly demonstrates the power of multidimensional single-molecule analysis in revealing key dynamics information of a molecular complex and visualizing rare events that are challenging by conventional ensemble methods. In fact, such a combinatorial approach has been used before in the studies of a number of complex systems (10–18) and this trend will certainly continue in the future. As mentioned above, Loparo et al. argue that even the productive polymerase exchange is more or less a passive process, which does not involve an active competition to gain access to the replisome by the old and new polymerase molecules. We believe it will be useful to investigate the nature of polymerase exchange directly, to rule out or take into account potential interactions between the two polymerase molecules. Such a study can be done by incorporating the capacity for single-molecule fluorescence resonance energy transfer, which has proved to be a versatile method for studying biological interactions.