The model bacteriophages T4 and T7 provide the simplest forms of the replisome. In these species, the replicative helicases, gp4 in T7 and gp41 in T4, are superfamily (SF) four helicases, which are characterized as homo-hexameric rings that employ a RecA-like motor domain to power translocation along single-stranded (ss) DNA in the 5′–3′ direction and concomitantly unwind duplex DNA by exclusion of the other strand. For T7, the gp4 translocation state is observed as a spiral staircase offset around single-stranded DNA (ssDNA) that structurally resembles one strand of A-form DNA, where each subunit contacts the backbone with a footprint of two nucleotides (nt) and with the nucleoside triphosphatase (NTPase) site holding an NTP at the interface between subunits (Figure 2A) (Gao et al., 2019). For T4, there are currently no structures available of gp41; however, sequence-based predictions of the structural motifs suggest a hexameric arrangement very close to that of gp4 helicase (Mueser et al., 2010).

These replicative helicases have different ways of interacting with the primase enzyme that promotes the synthesis of short RNA primers on the lagging strand. In the T7 replisome (Figure 3A), the primase is fused to the helicase and forms the N-terminal domain tier of gp4, but in the T4 replisome (Figure 3B), the primase is a separate protein (gp59) that binds to the gp41 helicase. Similarly, in each replisome the replicative helicase interacts with both the leading- and lagging-strand polymerases. The T7 gp4 helicase maintains a direct, physical connection to the gp5 polymerase. In T4, the gp43 polymerase is known to interact with the gp41 helicase but only transiently with lagging-strand replication likely occurring away from the fork. For a detailed review of T4 replication, see Benkovic and Spiering (2017) and for T7 replication see Kulczyk and Richardson (2016).

The E. coli replicative helicase, DnaB, is homologous to its bacteriophage counterparts, as it is also a homo-hexameric RecA-like helicase of the SF4 group. DnaB also uses its ATPase ability to translocate in the 5′–3′ direction on ssDNA and unwind double-stranded (ds) DNA. Similar to T7 gp4, the DnaB translocation state is also observed as a spiral staircase with each subunit holding a NTP at the subunit interface and in contact with 2 nt of the A-form-like DNA backbone (Figure 2B) (Itsathitphaisarn et al., 2012). It is important to note that this information is based on DnaB structures not from Gram-negative E. coli, but from the thermophilic Gram-positive species, Geobacillus stearothermophilus. However, the tertiary and quaternary structure is very similar to another, non-translocation state structure of DnaB in complex with DnaC from E. coli (Arias-Palomo et al., 2019).

Within the E. coli replisome (Figure 3C), DnaB interacts with 1–3 DnaG primase molecules via three binding sites on pairs of the helicase N-terminal domains. It is unclear if this interaction is constant or only transient during moments of primer synthesis and hand-off. Additionally, the DnaB helicase maintains a physical coupling to the polymerase III holoenzyme (leading and lagging Pol III cores, their associated β clamps, and the clamp loader complex) via the τ subunit of the clamp loader complex. It is not known where the τ subunit binds on the DnaB helicase, but it is expected that the strength of this interaction varies during replication as a means to direct primer hand-off and polymerase exchange (Monachino et al., 2020). For a detailed review of E. coli DNA replication, see Lewis et al. (2016).

The replicative helicase in the model eukaryote S. cerevisiae, is CMG, which consists of three parts: the Cdc45 activating factor, the MCM2–7 hetero-hexameric ring, and the hetero-tetrameric GINS. CMG is a SF6 helicase with as the main component, the MCM2–7 ring, which contains a AAA+ motor domain to power translocation along ssDNA and unwinding of double-stranded DNA (dsDNA). From structures of CMG bound to ssDNA, we see MCM2–7 form a partial spiral around A-form-like DNA, with the final two subunits closing the spiral (Figure 2C) (Georgescu et al., 2017). The C-terminal motor domain sits behind the N-terminal domain and pushes it in the 3′–5′ direction on ssDNA.

Due to this reversed directionality of CMG relative to the bacterial helicases, it translocates on the leading strand and excludes the lagging strand (Figure 3D). Therefore, the leading-strand polymerase Pol ε synthesizes DNA directly behind CMG and is expected to contact both GINS and the rear of MCM2–7. The lagging-strand polymerase Pol δ does not directly contact CMG. It is expected to either act behind the replisome or interact with CMG indirectly via the primase Pol α, or the Ctf4 organizing protein. The MTC complex also interacts with CMG to connect the helicase to multiple other replisome components. Furthermore, CMG has an essential yet ambiguous interaction with the Mcm10 firing factor which seems to enhance translocation and unwinding. A detailed review of the eukaryotic replisome can be found in Lewis and Costa (2020).

Ensemble biochemical studies have characterized replicative helicases and put forward the idea that these enzymes are universally poor motors when removed from their respective replisomes (Delagoutte and von Hippel, 2002; Delagoutte and von Hippek, 2003). The ensemble-averaging inherent in these methods, however, makes it challenging to uncover the precise molecular mechanisms underlying helicase activity. The principles that govern the rate of unwinding and the processivity of replicative helicases have not been fully elucidated, nor is it clear how the poor unwinding activity of isolated helicases is reconciled with the highly efficient unwinding occurring within replisomes. Single-molecule micro-manipulation techniques are very useful in this respect, as they can detect single events of unwinding with high sensitivity and offer the ability to apply force to the DNA to see how such mechanical manipulation influences enzymatic activity.

Several single-molecule studies were able to quantify individual unwinding events on tethered DNA templates using optical traps for the gp4 helicase (Johnson et al., 2007; Sun et al., 2011; Sun et al., 2015; Sun et al., 2018), and magnetic traps for the gp41 helicase (Lionnet et al., 2007; Manosas et al., 2010; Ribeck and Saleh, 2013), DnaB helicase (Ribeck et al., 2010) and CMG helicase (Burnham et al., 2019). All of these studies share an experimental design where a DNA template is tethered at one end to a glass slide and the other end to a bead that is manipulated by an optical or magnetic trap (Figures 1A,B; see The Single-Molecule Toolbox to Examine Helicases).

Each of the mentioned studies enabled detection of single helicases mediating individual unwinding events (Figures 4A,B). Each of the helicases show a relatively consistent unwinding rate interspersed with pauses, except for CMG, which was comparatively more sporadic (see further discussion of CMG in Eukaryotic (SF6) Helicase Mechanism). Unwinding is also occasionally followed by “rezipping” of the DNA template in the wake of the helicase. Rezipping presents as the slow, falling edge of bead movement and has been shown to be equivalent to the helicase translocation rate (Lionnet et al., 2007; Manosas et al., 2010). Interestingly, the measured unwinding rate varied among helicases, but each was consistently slower than the known corresponding replication rate (Table 1). Another commonality was the response to increased force applied to the occluded strand (>20 pN) where the unwinding rate (but not rezipping rate) increased exponentially (Figures 4C,D). Forces in this regime are expected to destabilize dsDNA, so therefore it is likely the free energy cost of unwinding at the DNA fork junction is proportionally reduced. Although seemingly removed from the conditions of the cell, this finding has two important implications for helicase unwinding mechanics that will be discussed below.

Firstly, these single-molecule studies confirmed that replicative helicases are inefficient at low forces, but rates comparable to those seen in replication can be obtained by applying a stronger force. Similarly, other single-molecule studies have shown that addition of the polymerase holoenzyme can improve the helicase unwinding rate and processivity (Manosas et al., 2012a; Manosas et al., 2012b). Therefore, a model starts to form of how polymerization acts to destabilize DNA at the fork to promote rapid helicase translocation and unwinding. Further discussion of the synergy between the helicase and polymerase are explored in Helicase-Polymerase Coupling Interactions.

The second important implication of the force-unwinding relationship relates to the thermodynamics of helicase-mediated unwinding. The process of ‘unzipping’ the next base pair is the rate-limiting step in the cycle of unwinding and can occur in two possible modes: either passive or active. For passive unwinding, ATP is only consumed for the helicase to translocate along ssDNA, and the DNA junction is advanced following thermal fraying of the proximal base pairs. For active unwinding, the energy from ATP hydrolysis by the helicase contributes to DNA junction destabilization as well as translocation. A dependence on force applied to assist duplex opening is a trait of the passive helicase unwinding model (Betterton and Julicher, 2003; Betterton and Julicher, 2005). Gp41, Gp4, DnaB, and CMG all exhibit this property—in distinct contrast to that of a known active helicase like RecQ (Manosas et al., 2010). Some of these helicases also demonstrate sensitivity to the AT content in the unwound DNA (Ribeck et al., 2010; Ribeck and Saleh, 2013; Syed et al., 2014; Schlierf et al., 2019), which is another trait of passive helicases. Intriguingly, for gp41, the measured force-rate curve is fit nicely by the passive model; however, for gp4 and DnaB, the results deviate from the model at high forces. Thus, it is tempting to argue that these helicases must somehow contribute to DNA junction destabilization and are thus “weakly active”.

A “weakly active” helicase is an enzymatically interesting concept; however, the available data do not unequivocally support this conclusion and its implications for replicative helicases. We should also question the robustness of the passive unwinding model. The model is extremely sensitive to the value of step size and slippage frequency—parameters that are both very difficult to measure. Notably, for gp4 the data fit well with different parameter sets, which actually predict different unwinding mechanisms (Manosas et al., 2010; Chakrabarti et al., 2019). As a consequence, the topic of passive versus active mechanism remains controversial, with the literature rife with conflicting statements about the true unwinding mechanism based on different methods. It is possible that all these replicative helicases are passive, and thus far we have not been able to identify the proper model. Alternatively, some of these helicases could be partially active, but we do not currently possess techniques with the necessary discriminatory power to confirm this hypothesis. In the future we should look for more comprehensive theoretical modelling (see Chakrabarti et al., 2019) and further experimental studies with higher throughput to verify these predictions.

Unlike monomeric or dimeric helicases, the replicative helicases are all hexameric rings (O'Donnell and Li, 2018) and therefore require a great deal of coordination among subunits to achieve translocation. Through the power of single-molecule methods like smFRET and trapping assays, we are able to observe individual unwinding events mediated by single helicases in real-time with the resolution approaching individual base pairs. From these experiments it is possible to investigate the coordination of hexameric subunits by quantifying and comparing intra- and inter-molecular dynamics during unwinding.

It is due to the use of single-molecule methods that we have observed helicase slippage. By manipulating gp4 at the single-molecule level using an optical trap, Sun et al. (2011) detected that the helicase mostly maintains a constant unwinding rate but occasionally slips backwards on the DNA. This helicase slippage is a potential indicator of desynchronized subunits. The authors also found that gp4 slippage only occurs in the presence of ATP, and not dTTP, the latter being the preferred nucleotide during replication (Matson and Richardson, 1983). Therefore, it is conceivable that ATP-induced slippage is a backup mechanism to keep the helicase close to the polymerase if the polymerase ever slows or stalls when the dNTP pool is depleted and decoupling between the enzymes occurs.

Slippage is also observed in a single-molecule trapping study of T4 gp41 (Manosas et al., 2012a) and a smFRET study of G40P, the DnaB-like helicase of phage SPP1 (Schlierf et al., 2019). Interestingly, these helicases both demonstrated a higher frequency of slippage at regions of DNA with a high GC content (Figures 5A,B). This aligns with the expectation that replicative helicases passively unzip DNA (as discussed in Active Versus Passive Helicases). In this passive model, GC base pairs present a higher free energy barrier for fraying and thus greater chance of failure and subsequent slipping. It is likely that slippage is attributable to either the simultaneous unbinding of all subunits from DNA or a cascading effect after one subunit unbinds. Interestingly, it has been demonstrated that this slippage effect is negated by the presence of the polymerase (Manosas et al., 2012a) or primase (Figure 5B) (Schlierf et al., 2019), suggesting that slippage only ever occurs when the helicase is decoupled from the replisome (see Replisome Coupling for further discussion).

There was no detection of CMG helicase slippage when observing unwinding events at the single-molecule level using magnetic traps (Figure 4B) (Burnham et al., 2019). There are several possible explanations for this observation. CMG does interact with several other replisome components, such as Pol ε, Mcm10, and MTC, that could potentially inhibit slippage (reviewed in Lewis and Costa, 2020), but they were not present in this assay. It is possible that the GC content of the DNA template of this assay did not provoke slippage. Another alternative is that the MCM2–7 hetero-hexamer arranges the different subunits around DNA in a way that prevents slipping. However, we do not understand the unwinding mechanism of CMG well enough to pass this assessment (see Eukaryotic (SF6) Helicase Mechanism). Alternatively, in structures of CMG, the Cdc45 subunit is in contact with both the MCM2–7 ring and DNA. It has therefore been suggested that this protein acts as an internal brake within the helicase to prevent slippage events (Petojevic et al., 2015). High-resolution single-molecule FRET might be applied in the future to observe CMG activity on DNA templates (Figure 1C; see Step Size), to see if the helicase responds differently to variations in the GC content of the template.

During unwinding, helicases separate base pairs in discrete, repeated events known as steps. Thus, it is important to determine the factors that contribute to the stepping action and step size of a helicase. Step size can be further defined as either the “physical” or “kinetic” step size, where the former is the physical distance (in nucleotides) traveled per NTP hydrolyzed, and the latter is the distance between two rate-limiting transitions.

In all structures of the replicative helicases bound to ssDNA (Figure 2), each helicase subunit contacts the backbone of two nucleotides. These structures imply that each of these helicases move with a physical step size of 2 bp per ATP cycle. In contrast, early single-turnover ensemble measurements of the kinetic step size of gp4 resulted in an estimate of ∼10 bp (Jeong et al., 2004). The lab of Taekjip Ha has developed single-molecule assays based on FRET to detect helicase unwinding events with a structural resolution of ∼3 bp (Figure 1C; see The Single-Molecule Toolbox to Examine Helicases) (Deniz et al., 1999; Ha et al., 2002). They have since applied this assay to a number of helicases, including gp4 (Syed et al., 2014) and DnaB-like, phage SPP1 G40P (Schlierf et al., 2019). They were able to directly detect small steps of 2–3 bp for both helicases. Interestingly, further analysis of the single-molecule kinetics showed that the dwell time between steps was best described by a gamma distribution rather than an exponential one. Such a fit suggests that there are several rate-limiting kinetic steps hidden within bursts of 2–3 bp of unwinding.

This theory on stepping action was recently proven by direct observation. Lin et al. (2017) were able to further enhance the resolution of the single-molecule FRET unwinding assay with the addition of a nanotensioner. A nanotensioner is a short DNA duplex designed to apply force to both ends of the unwinding template overhangs (Figure 5C). As a result, the fluorophores on the overhangs are stabilized and the FRET resolution is improved to <1 bp (Lin et al., 2017). When used to study gp4, the nanotensioner-enhanced FRET unwinding assay revealed that the helicase took a variety of step sizes between 1–4 bp, with 2 bp being the most common (Figure 4D) (Ma et al., 2020). A possible explanation for the variation in step size is to consider that these hexameric helicases sample a hierarchy of steps where part of the elastic energy from a prior step can contribute to the next step. In their study, Ma et al. also found that gp4 stalled at abasic lesions, where it shuffled 1 bp back and forth and then occasionally jumped past the lesion. The ability to sample different step sizes and ‘leapfrog’ lesions could be a potential mechanism to confer robustness by ensuring the helicase, and by extension the replisome, acts processively to duplicate DNA.

The only replicative helicase not discussed here is CMG. Currently our knowledge of the CMG helicase stepping mechanism is severely limited. Structural data shows the MCM subunits each in contact with 2 nucleotides (Figure 2C), yet the step size could still vary (Georgescu et al., 2017). Furthermore, this and other CMG structures (Abid Ali et al., 2016; Yuan et al., 2016) show CMG arranged in a different quaternary state compared to gp4 and DnaB, so it is challenging to derive any information from the prokaryotic model to predict the likely nucleoprotein stepping dynamics of eukaryotic CMG. Single-molecule assays with high spatial-temporal resolution seem the best path forward to help elucidate the step size of CMG.

Structural studies have arguably made the greatest contribution to our knowledge of helicase unwinding mechanisms. Atomic-level visualization of helicase structures gives us an idea of the different static architectures of unwinding; however, recent single-molecule studies have been able to report on the intricate dynamics that occur in between these states. The following sub-sections compile our current understanding of unwinding mechanisms and highlight the progress derived from single-molecule studies.

Loading of the replicative helicase is the key step in initiating DNA replication. This process begins with initiator proteins recognizing origins of replication and loading replicative helicases in specific locations, allowing initiation of replication in a bidirectional manner at the correct time. Between species, distinctly different players are involved in initiation, however in each system the process is always tightly controlled. Genetic studies in E. coli have shown that if some of the initiation factors are suppressed, unregulated and untimely initiation can occur—often with lethal consequence (Charbon et al., 2018). Ensemble-averaging studies have made tremendous headway to characterize the proteins involved in initiation, but single-molecule techniques hold a greater resolving power capable of identifying the initiation assembly order and potential protein intermediates along the way.

The first example of single-molecule investigation of helicase loading during replication initiation was conducted by Benkovic and coworkers. Their work was able to identify the order of key events during T4 initiation. This process is still ambiguous because it is debated whether the gp41 helicase and gp43 polymerase maintain a physical coupling in replication and thus if their initiation is synchronized (Spacciapoli and Nossal, 1994; Dong et al., 1996; Delagoutte and von Hippel, 2001) (see Helicase-Polymerase Coupling Interactions). Previous work has shown that the gp59 helicase loader has strong affinity for ssDNA (Jones et al., 2004). It has been suggested that gp59 along with the gp32 single-stranded DNA-binding protein binds the fork first and then recruits the gp41 helicase. The use of single-molecule colocalization and FRET has demonstrated that gp59-gp32 first binds and locks the gp43 polymerase on ssDNA. Addition of the gp41 helicase results in its loading, which then unlocks the polymerase (Xi et al., 2005a; Xi et al., 2005b; Zhang et al., 2005). Therefore, it seems that the gp59 helicase loader plays a critical role to orchestrate T4 replication initiation and synchronize unwinding and polymerization.

Single-molecule methods have also been useful to untangle the intricate process of replication initiation in eukaryotes (reviewed in detail by Lewis and Costa, 2020). At present, we understand the first stage as “origin licensing”, which is temporally confined to the G1 phase of the cell cycle. First, the origin recognition complex (ORC) binds directly to one of the many origin DNA sequences. Next the accessory initiator proteins Cdc6 and Cdt1 recognize, bind and thus become a “marker” of the helicase loading loci. These two proteins then facilitate the loading of two MCM2–7 rings in a head-to-head, double hexamer orientation. When the cell reaches S phase, the second stage described as “origin firing” begins, in which MCM2–7 is converted into a complete CMG helicase with the addition of the Cdc45 and GINS proteins, and finally processive unwinding is activated by the firing factor, Mcm10.

This initiation pathway is highly concerted; however, many features remain ill-defined, with one of the key questions being whether the initial loading of the two helicases occurs simultaneously or sequentially. Ticau et al. answered this question in their single-molecule study when they reconstituted the initiation process in vitro and observed loading events using real-time colocalization methods (Figure 1D; see The Single-Molecule Toolbox to Examine Helicases) with fluorescently labeled DNA and MCM2–7 hexamers. These assays were able to distinguish sequential fluorescence steps corresponding to MCM2–7 hexamers loading one after the other (Figures 6A,B) (Ticau et al., 2015). Further colocalization experiments showed that each hexamer loading was correlated with the binding of a different set of Cdc6 and Cdt1 proteins (Cdt1 example shown in Figure 6B). These results were able to demonstrate sequential MCM2–7 loading and also suggest the second hexamer loads in a process distinct from the first. Several years later, the same research group built on their original assay by labeling different MCM2–7 subunits with a FRET pair and monitoring ring closure during loading (Ticau et al., 2017). They observed both donor and acceptor fluorescence as an indicator of binding, as well as FRET as a measure of ring opening and closing (Figures 6C,D). The results indicated that as each MCM2–7 hexamer loads, it opens its ring briefly and then closes as it encircles DNA (Figure 5D). This study also identified that ring closure is tightly correlated with ATP hydrolysis and departure of the associated Cdt1 (Ticau et al., 2017).

Other single-molecule studies have been able to corroborate these observations. Specifically, single-molecule use of correlative fluorescence and force spectroscopy made it possible to detect a gate in CMG that opens when the helicase transitions from translocating ssDNA to dsDNA (Wasserman et al., 2019). This study also identified that this gate is distinct from the ring opening mechanism and is possibly how CMG exits the double-hexamer structure during origin firing. Furthermore, Champasa et al. (2019) identified the key residues essential for double-hexamer formation using a combination of mutations and smFRET. In a separate study, Duzdevich et al. (2015) employed a DNA curtain assay to observe hexamer loading within a larger replication initiation pathway. Collectively, these results indicate the mechanism of how replication is controlled to only “fire” once in a bidirectional manner. They also highlight the degree to which replication initiation is a highly coordinated process in eukaryotes.

Interestingly, no single-molecule studies have been done on helicase loading in bacteria. This process has been studied extensively via ensemble biochemical methods and structural analysis (reviewed by Chodavarapu and Kaguni, 2016), yet single-molecule examination could be very useful to clarify the replisome assembly pathway at oriC and pinpoint the activation of the DnaB helicase.

All replication complexes need to be highly processive in DNA replication to fully duplicate the long chromosomal molecules. Essentially, this means that the main enzymes of replication, the polymerase and helicase, need to sustain their activity for the entirety of the replication process. Especially for E. coli, this expectation of high replisome processivity is based on the known genome size (4.6 Mbp) and its duplication time (∼40 min) (Chandler et al., 1975), coupled with the necessity for only two oppositely-traveling replisomes, but yet the relatively infrequent rate of replication collapse (once in every 5 generations) (Maisnier-Patin et al., 2001). Along with the observation of a relatively stable replication complex, this led to a model of replication where the required high processivity of the replisome was strongly linked to its high stability (Beattie and Reyes-Lamothe, 2015).

In contrast to this model however, observations of DNA replication at the single-molecule level revealed that the replisome is not a stable entity but is in fact highly dynamic with components rapidly exchanging in and out. This exchange phenomenon was first hypothesized and detected within the T7 replisome (Loparo et al., 2011; Geertsema et al., 2014), and has since been identified in bacteria (Liao et al., 2016; Beattie et al., 2017; Lewis et al., 2017; Li et al., 2019; Spenkelink et al., 2019; Dubiel et al., 2020) (Figure 7A) and most recently, in eukaryotes (Kapadia et al., 2020; Lewis et al., 2020) (Figure 7B).

In particular, the single-molecule application of FRAP and FLIP have made it possible to measure the kinetics of this exchange process (Figures 1E,F; see The Single-Molecule Toolbox to Examine Helicases). In E. coli, FRAP applied in vivo, at the single-molecule level, to fluorescent versions of the replicative Pol III identified exchange to occur every few seconds (Beattie et al., 2017). Parallel in vitro FRAP experiments were able to detect individual E. coli Pol III exchange events during in vitro rolling-circle DNA replication and found the process to occur in a manner dependent on the concentration of free polymerase (Lewis et al., 2017). A similar effect was identified in S. cerevisiae, where again single-molecule FRAP was applied in vivo to replisome-bound polymerases (Kapadia et al., 2020) and in vitro to polymerases within a replication assay based on linear templates (Lewis et al., 2020); the in vitro study demonstrated concentration-dependent polymerase exchange — albeit at a slower rate than observed in E. coli.

FLIP is often used to complement FRAP as it measures the loss of fluorescence when a protein exchanges for an unlabeled one. In the cases where FLIP was also used in these studies, it did indeed confirm the results of FRAP (Beattie et al., 2017; Kapadia et al., 2020).

As this dynamic replisome model becomes more solidified in the literature, it raises the question: if the replisome is not stable, how does the complex sustain processive replication? It seems the same single-molecule methods also provide the means to answer this question. Through single-molecule FRAP and FLIP, it has been shown that unlike the polymerases, the replicative helicase exchanges rarely, if at all (Figure 7A). In E. coli, in vivo FRAP measurements showed fluorescent DnaB never fully recovered its signal, indicative of very stably incorporated helicases (Figure 7C) (Beattie et al., 2017). This observation was corroborated by in vitro real-time observations of fluorescent DnaB molecules during rolling-circle replication (Spinks et al., 2021). Here, the DnaB helicase displayed ambiguous recovery in FRAP, but through the use of FLIP, it was shown that once incorporated into the replisome, DnaB is impervious to challenge by a large excess of DnaB molecules in solution (Figure 7E). Comparable observations have been made in S. cerevisiae, where fluorescent CMG demonstrated long residence times and low FRAP recovery in vivo (Figure 7D) (Kapadia et al., 2020). Also, examination of fluorescent CMG with FLIP during in vitro reconstituted replication established that the helicase is unaffected by the presence of excess CMG (Figure 7F) (Lewis et al., 2020). The stability of the phage helicases, gp41 and gp4, have yet to be assessed at the single-molecule level. Yet, it is expected to be consistent with the E. coli data owing to their similar replisome architecture.

Collectively, these results suggest that even in different phyla, the replicative helicase very rarely exchanges during DNA replication. Instead, after one helicase is loaded and replication is initiated, it is likely that a single helicase is maintained for the entirety of the replication process. In this sense, the replicative helicase becomes the anchor of the replisome, ensuring processive replication and providing a constant binding site for other exchanging proteins (Figures 7A,B). Furthermore, this high degree of helicase stability is likely the reason for the intricate replication restart pathways that exist across all life to reload the replicative helicase in cases where it has dissociated. It would be interesting to see future work use this kind of single-molecule approach to observe stalled replication forks and examine replisome dynamics in those events.

In all replisomes, the primary responsibility of the replicative helicase is to unwind double-stranded parental DNA into two template strands for copying. Unlike non-replicative helicases, these replisome-bound helicases also seem to have a secondary role to act as anchor points for the larger multi-protein complex (as discussed in Stability During Replication). Interestingly, the interactions between the replicative helicase and other replisomal proteins also appear to enhance helicase activity (as discussed in Active Versus Passive Helicases). Observations of replicative helicase activity at the single-molecule level has helped tease out the intricacies of the coupling interactions the replicative helicase has with the replisomal polymerase, primase and single-stranded DNA-binding proteins (SSBs).