In the simplest sense, expressing a gene means manufacturing its corresponding protein, and this multilayered process has two major steps. Mutations that happen during Transcription and Translation. What happens if there is a mistake mutation in the DNA code? Proteins that fold improperly may also impact the health of the cell regardless of the function of the protein.
When proteins fail to fold into their functional state, the resulting misfolded proteins can be contorted into shapes that are unfavorable to the crowded cellular environment. Errors during translation elongation that result in incorporation of an incorrect amino acid, frameshifting see Glossary , readthrough of stop codons, or premature termination can produce proteins that deviate from the encoded amino acid sequence.
The most common type of transcription error is a C to U base substitution and transitions occur more frequently than transversion epimutation events, as has been found for spontaneous mutation [9, 11], therefore RNA polymerase base misincorporations appear to resemble DNA polymerase base misincorporations.
When Replication Errors Become Mutations. Incorrectly paired nucleotides that still remain following mismatch repair become permanent mutations after the next cell division. This is because once such mistakes are established, the cell no longer recognizes them as errors.
Begin typing your search term above and press enter to search. Press ESC to cancel. Skip to content Home Philosophy What is the difference between helicase and polymerase? Ben Davis May 19, Errors shown are standard deviations from average of 2—5 experiments. Taken together, these results indicate that T7 DNAP binds to three template-bases downstream of primer-end and melts two base pairs. However, T7 helicase increases the fluorescence of 2-AP at the fork junction, but not the second base pair from the junction, irrespective of the gap size between the primer end and fork junction Figure 5E—H —Red bars.
Thus, unlike T7 DNAP that melts two base pairs upon binding to the replication fork substrate, T7 helicase melts only the junction base pair. However, T7 helicase melts the junction base pair even when the primer-end is separated from the fork junction by more than one nucleotide Figure 5—figure supplement 5A—C compare to Figure 5—figure supplement 2.
These results indicate that T7 helicase follows the fork junction and is not influenced by the position of the primer-end. Interestingly, SSB has no effect on the helicase catalyzed melting of the fork junction Figure 5—figure supplement 5D. This is consistent with the observation that SSB does not stimulate the unwinding rates of the helicase Donmez and Patel, The 2-AP fluorescence intensity at steady state measures the equilibrium distribution of melted and annealed states of the junction base-pair.
The small increase in fluorescence intensity with the isolated helicase and DNAP suggests that each enzyme shifts the equilibrium only moderately to the base-pair melted state. On the other hand, the striking increase in fluorescence intensity with the combined enzymes indicates that together the two enzymes shift the equilibrium strongly toward the base-pair melted state.
Interestingly, the combined effect of helicase and DNAP on base pairs melting is greater than the sum, which indicates synergism in DNA melting. This synergism depends on the number of nucleotides between the primer-end and fork junction. Synergistic melting of the base pair is observed only when there is no gap or one nucleotide gap between the primer-end DNAP-binding site and fork junction helicase-binding site Figure 5E—G. Synergistic melting is not observed when there are two nucleotides between the primer-end and fork junction Figure 5H.
The results also demonstrate that a replication fork with two ssDNA template-bases between the primer-end and fork junction can stably accommodate both enzymes of the T7 replisome. Therefore, this study defines the specific positions of helicase and DNAP at the replication fork junction with single-base resolution to create a structural model of the replisome Figure 6 that forms the basis for understanding how the helicase and DNAP mutually stimulate each other's activities as discussed below.
This model explains the one-nucleotide step size where the combined enzymes translocate by one nucleotide for every dTTP hydrolyzed and nucleotide incorporated Pandey and Patel, This was unexpected, because DNA synthesis occurs in steps of one nucleotide, which would require unwinding of only one base pair at a time.
Two or more template-bases in the downstream template-binding pocket have been observed in crystal structures of other DNAPs as well Doublie et al. We show that T7 helicase on its own can also melt the junction base pair, but it melts only one base pair and follows the fork junction. Comparing DNA melting by isolated and combined enzymes reveals that the junction base pair is melted only partially by the isolated enzymes Figure 5E—H. This explains the slow and GC sensitive unwinding rates of the isolated enzymes in our kinetic experiments Figures 1C, 2C.
The junction base pair is melted more efficiently by the combined enzymes, explaining fast and GC-independent rates of the combined enzymes Figure 3B.
We observed that the combined enzymes melted two base pairs upon binding to the fork DNA. We propose that this occurs initially to establish the catalytically competent structure of the replication fork—T7 DNAP melts two base pairs and binds two ssDNA nucleotides in its template-binding pocket, which positions the helicase two nucleotides ahead at the fork junction, as shown in Figure 6. We propose that during active leading strand synthesis, the enzymes melt only one base pair at the fork junction at a time consistent with the one nucleotide chemical step size of the combined enzymes Pandey and Patel, Based on our results, we propose that the DNAP by itself is not efficient at preventing junction base pair reannealing, and this unfavorable equilibrium constant for DNA melting destabilizes the post-translocated state of DNAP and competes with incoming dNTP binding.
When helicase is present at the fork junction, it helps both unwind and trap the junction bases. The helicase by itself is not efficient at unwinding the fork junction.
However, the associated DNAP by providing an unwound base to the helicase at the fork junction facilitates the base-capture step and drives the reactions of dTTP binding-hydrolysis-product release around the helicase ring, and the outcome is an increase in the unwinding k cat. The combined binding energy of the two enzymes bound to opposite strands is sufficient to keep the unwound bases from reannealing, explaining the fast and GC-independent unwinding rates of helicase-DNAP.
Interestingly, cooperative and enhanced efficiency of base pair melting is observed only when the helicase and DNAP are within one nucleotide distance from each other. In most cases, helicase is coupled physically to the DNAP, either directly as in the case of T7 replication system or indirectly through accessory proteins Kim et al. Some of these interactions aid in the assembly of the replisome Zhang et al. One can imagine situations where flexible positioning is needed when one or the other enzyme pauses or stalls during leading strand synthesis.
Our investigation of such situations reveals that when DNAP stalls or is the slower motor, the helicase becomes functionally uncoupled and outruns the DNAP by unwinding the replication fork at the unstimulated rates. Similar behaviors were observed in other replisome studies as well Byun et al. Whether the functionally uncoupled helicase remains physically coupled to the DNAP remains unknown.
Interestingly, when the helicase slows down, the two enzymes remain functionally coupled as evident from the low dNTPs K m and that the DNAP does not outrun the helicase.
In this case, the combined enzymes unwind the DNA with the stimulated rate of the helicase. We propose that this is because SSB cannot trap the junction bases coordinately with DNA synthesis in the manner that T7 helicase does during leading strand synthesis.
The replicative helicase is a central player in coordinating leading and lagging strand synthesis Pandey et al. The interdependency between helicase and DNAP assures that the DNA is not unwound in an uncoupled manner leading to disruption in the coordinated synthesis of the two strands. Our studies suggest that the leading edges of the T7 helicase and T7 DNAP are close together at the fork junction and this conformation is important for functional coupling of unwinding and synthesis reactions and preventing DNA reannealing.
This model of the replication fork is likely to be generally applicable to replisomes of prokaryotes as most show functional coupling between helicase and DNAP Patel et al.
In contrast to prokaryotic replisomes, the replicative helicase of eukaryotes and archaea binds to the same strand as the DNAP O'Donnell et al. In this case, both helicase and DNAP cannot be close to the fork junction, and there must be other mechanisms to functionally couple the two activities and prevent junction base pair reannealing.
The DNA sequences are provided in Supplementary file 1. Thioredoxin was purchased from Sigma St. Louis, MO. The sample was excited at nm 2 mm slit width , and emission was measured at nm 6 mm slit width. The buffer contained 50 mM Tris Cl pH 7. The observed fluorescence was corrected for buffer and protein bound to unlabeled replication fork substrate. The proteins absorb minimally at the emission wavelength, and hence, the inner filter effect was negligible.
The DNA unwinding kinetics were fit to the n -step model Ali and Lohman, using gfit and model [unwinding. Unwinding is modeled as a multistep process with equal step-size s and rate constant k i that are estimated from fittings as described previously Pandey et al.
More information about the fitting is provided in the Appendix —Methods section. The average unwinding rates were plotted against dNTP concentration and fit to the hyperbolic equation to obtain k cat and K m values.
The DNA unwinding kinetics show an initial time lag followed by an increase in fluorescence in two phases, a fast phase and a slow phase Figure 1—figure supplement 2A. Increasing E. Therefore, the unwinding rates by T7 DNAP were estimated by fitting the fast phase to the stepping model.
The fast phase rate matches closely with the strand displacement rate obtained from the gel-based assays Pandey and Patel, The K 1 is the equilibrium constant for the base-capture step. This model predicts that k cat remains constant with increasing GC content, which is not what we observed Figure 2D.
It is difficult to directly measure the apparent K d, dTTP of the leading subunit of the hexameric helicase that has captured the DNA base at the fork junction. The pre-steady state kinetics of dTTP hydrolysis was measured as described earlier Kim et al.
The DNA-unwinding kinetics were fit to the n -step model Ali and Lohman, using gfit and model [unwinding. To account for heterogeneity, the model calculates the sum of N unwinding processes. The stepping kinetics are described by the following equations. F is the fraction of DNA substrates molecules that have completely undergone strand displacement synthesis, A i is the amplitude and t is reaction time. The number of steps, n , is given by. L m is negligible under stopped flow assay conditions where there is no lag between reaction termination and data observation Donmez and Patel, and hence set to 0.
Best fits were obtained assuming unwinding by more than one population with identical step size, but different stepping rates. An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown.
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Your article has been favorably evaluated by John Kuriyan Senior editor and three reviewers, one of whom is a member of our Board of Reviewing Editors.
The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. This manuscript furthers our understanding of the functional coupling between helicase and DNA polymerase for rapid and efficient DNA replication. The authors varied the cofactor concentration in a series of unwinding assays to determine kinetic parameters highlighting the stimulatory effect of the two enzymes to increase the efficiency of the base-capture step of the other enzyme by influencing the melting of the junction base-pair of the DNA fork.
In line with the track record from this group the experiments are of high quality, yet some points should be addressed. Most comments require only clarification of the text, but some additional experiments, which should not take much time, are needed.
In the past, this group has made use of different lengths of dsDNA to determine the kinetic parameters. This becomes important especially with the DNAP data collected in the presence of SSB where, in addition to a lag, the traces show a clear biphasic behavior. The authors chose to analyze only the first phase. How is this signal amplitude correlated to the fraction of dsDNA unwound? This parameter appears to be in Equations The authors offer no proof or explanation that the latter can simply be ignored as they have done.
Our detailed study of the holoenzymes presented elsewhere 14 demonstrates that at low applied forces the DNA fork regression caused polymerization by the holoenzymes to stall. In this situation, the exo conformation i. The polymerase stalling induced by the fork regression pressure and the consequent pol-exo equilibrium switch is responsible for the low efficiency of strand displacement DNA synthesis exhibited by these replicative polymerases Moreover, in contrast to the T4 or T7 holoenzyme, its DNA synthesis rate was not affected much by the presence of the DNA duplex upstream of the polymerase.
Along the range of forces studied, the strand displacement synthesis rate nearly coincided with the primer extension rate, indicating that this polymerase is extremely efficient at performing strand displacement synthesis. We have previously demonstrated the ability of the T4 helicase to unwind a DNA hairpin and continue translocating on the ssDNA followed by reannealing of the hairpin behind the helicase Figure 2 A 17 , In contrast, the gp41 unwinding activity was strongly force-dependent with the unwinding rate increasing almost exponentially with the applied force.
The helicase also occasionally paused at regions of high GC content during its unwinding activity demonstrating that the DNA sequence affected the helicase activity Supplementary Figure S2B. This strong force and sequence dependence revealed that gp41 relies, at least partially, on the thermal fraying of base pairs in order to unwind the DNA fork, which has been associated with the mainly passive character of this helicase 17 , We have made the new observation of tens to hundreds of base pairs suddenly reannealing during an unwinding phase Figure 2 B.
The unwinding activity immediately before and after these rapid reannealing events observed at low helicase concentration so that one unwinding event was observed every tens of minutes supports the conclusion that the same helicase complex remained bound to the DNA.
We propose that these rapid reannealing events were generated by the regression pressure of the DNA fork pushing the helicase backwards while attempting to unwind the DNA hairpin.
We refer to this phenomenon as helicase slippage. Helicase slippage was initially introduced by Delagoutte and von Hippel 5 as ATP hydrolysis events that resulted from a helicase advancing by less than one full translocation step size e. Here, we extend this concept to include the helicase sliding backwards.
The dependence on the ATP concentration suggests that slippage events were related to a low-affinity-for-DNA state of the helicase e. Helicase slippage has recently been observed for the T7 helicase 21 and such low-affinity-for-DNA states have been proposed for the T7 helicase as part of its ATP-hydrolysis cycle Our results suggest that the fork regression pressure acting on the low-affinity-for-DNA state of the helicase not only reduced its forward motion as previously proposed 5 , 20 , but also induced the helicase to slide backwards.
Overall, we conclude that the helicase activity at low force or in the absence of an external force is not only limited by its passive character, but also by its tendency to slip backwards. A replisome is comprised of many proteins working together to replicate the DNA; consequently, some of these other proteins might play a role in modulating the activity of the holoenzyme and helicase.
In particular, gp32 is the single-stranded DNA-binding protein in T4 responsible for coating the ssDNA as it is unwound making it essential for lagging-strand synthesis 23 ; is known to interact directly with several replisome proteins including gp43 polymerase, gp45 clamp, gp59 helicase loader and gp61 primase 24— Indeed, addition of gp32 to our wt and exo T4 holoenzyme strand displacement synthesis assay shows the almost complete suppression of the processive exonuclease activity and a significant decrease in the holoenzyme stalling behaviour Supplementary Figure S3A.
In addition, the T4 helicase unwinding rate computed from the sections of the experimental traces without detected slippage was increased by a factor of 1. A pause in synthesis occurred when the two enzymes collided head-to-head on the DNA, an artefact created by our hairpin substrate design; eventually the synthesis of the remaining part of the hairpin substrate was completed in the primer extension mode.
These results demonstrate that while the coupling between the holoenzyme and helicase produced very efficient stimulation of their separate enzyme activities, any direct physical interaction between the holoenzyme and helicase was weak and could be broken under conditions where the helicase moved faster than the holoenzyme. Homologous coupling of the T4 holoenzyme and helicase.
Initially, the holoenzyme loads and begins DNA synthesis in the strand displacement mode. When the helicase loads, it unwinds the DNA faster than the holoenzyme synthesizes the DNA, passes the loop apex and begins translocating along the leading strand, and collides with the holoenzyme orange.
C The rate of helicase unwinding, wt and exo holoenzyme strand displacement synthesis and coupled replication in the absence and presence of ssDNA-binding protein are shown as a function of the applied force. Supplementary Figure S4 shows how V rep , and varied with the ATP or dNTP concentration demonstrating that both enzymes modulated the replication rate with the maximum rate being limited by the slowest enzyme.
Previous studies have shown that heterologous combinations of holoenzymes and helicases from T4 and T7 could perform coupled leading-strand DNA synthesis 5 , 6. When testing the pairing of the T7 holoenzyme and the T4 helicase, results similar to the T4 homologous system were obtained: rapid and processive coupled leading-strand DNA synthesis at low forces and uncoupled holoenzyme and helicase activities at high forces.
This work has attempted to understand the mechanism of functional coupling between replicative holoenzyme complexes responsible for DNA synthesis and helicases responsible for unwinding duplex DNA to produce rapid and processive strand displacement synthesis which mimics leading-strand DNA synthesis in a replisome.
By using the T4 bacteriophage as a model system, we first tested the activity of the individual enzymes and found that their unwinding or strand displacement activities were very low when not assisted by force. In stark contrast, the T4 homologous coupled system was very efficient at low forces and DNA synthesis advanced at the maximum rate without exhibiting fork regression or pauses.
We modelled the individual enzyme behaviours and propose a collaborative model to best explain the homologous and heterologous coupling results under various experimental conditions. A passive motor protein inefficient at reducing the barrier must rely on the transient opening fluctuations of the upstream DNA duplex and therefore would move slower on dsDNA than on ssDNA.
A detailed description of the modelling is given in the Supplementary Materials. Model for passive and active enzymes. Modelling of the unwinding activity, excluding helicase slippage, best characterized the T4 helicase as a mainly passive 0.
The implication is that the T4 helicase must rely on the transient opening of the base pair at a fork in order to step forward due to its inefficiency at destabilizing the upstream DNA duplex. Modelling of the T4 and T7 holoenzyme polymerization activity, excluding pausing and exonuclease activity, best characterized the holoenzymes as strongly active motors 1. This characterization might seem at odds with the processive exonuclease activity exhibited by these holoenzymes in a strand displacement configuration.
By excluding holoenzyme pausing and exonuclease activity from our analysis, we effectively modelled the behaviour of the holoenzymes only in the pol conformation and excluded the inactive or stalled I intermediate and exo conformations The implication is that the T4 and T7 holoenzymes are active motor proteins efficient at destabilizing the upstream DNA duplex if the pol-exo equilibrium can be shifted towards the pol conformation.
Crystal structures of RB69 a structural and functional T4 homolog and other polymerases bound to a primed DNA molecule 30 , 31 give some insight into the possible mechanism for base pair destabilization.
This sharp kink in the template strand produces a stress, due to the intrinsic rigidity of the ssDNA, which could propagate along the template strand destabilizing the upstream DNA duplex. The most obvious mechanism to explain the dramatic increase in DNA synthesis and unwinding rates and processivity of the coupled reaction is a direct protein—protein interaction that leads to an allosteric effect stimulating one or both of the enzyme activities.
While several groups have searched, no physical interaction between the gp43 polymerase and the gp41 helicase has been detected by analytical ultracentrifugation 5 or by protein—protein cross-linking The T7 holoenzyme and helicase exhibit similar rapid and processive coupled strand displacement synthesis and share a direct protein—protein interaction through patches of basic residues on the polymerase and the C-terminal tail of the helicase 33 , 34 ; however, Stano et al.
Moreover, the presence of heterologous coupling between the T4 and T7 enzymes and the force induced uncoupling observed in our assays also argues against a strong specific binding as the mechanism for coupling, but does not rule out a non-specific association of the involved proteins. The emergence of single-molecule techniques to monitor the activity of individual enzymes has permitted us to obtain direct evidence of the T4 helicase slipping backwards tens to hundreds of base pairs due to the regression pressure of the DNA hairpin substrate.
Therefore, the modest stimulation of the unwinding rate, still well below the ssDNA translocation rate of the T4 helicase or the coupled strand displacement rate, indicates that preventing helicase slippage alone cannot account for the processivity and rate enhancement of the coupled holoenzyme and helicase. In agreement with this scenario, we found that the synthesis rate of the coupling reaction is controlled by the holoenzyme polymerization rate.
However, our results also point out the importance of the helicase at the replication fork, not only to provide ssDNA template for the holoenzyme, but also to relieve the fork regression pressure on the holoenzyme preventing holoenzyme stalling. The collaborative coupling model proposed here is based on the idea that the two individual enzymes assist each other to maximize the efficiency of their combined activity.
To characterize coupled leading-strand DNA synthesis, we combined the separate models used to describe helicase unwinding and holoenzyme polymerization.
Because of the random assembly of the enzymes on the DNA fork, the relative position of the two enzymes was not imposed a priori and both possibilities were evaluated. However, we presumed that the presence of the DNA fork affected only the enzyme in the leading position. In other words, the DNA fork only represented an extra energetic barrier for the advance of the leading enzyme, while the trailing enzyme could advance freely on ssDNA. If the helicase were ahead of the holoenzyme, then the helicase was allowed to move forward and backwards in response to the fork pressure, but the holoenzyme was considered to remain in the active pol state.
In the opposite situation, when the holoenzyme was in the leading position transitions from the pol state to the exo state of the holoenzyme were allowed. Kept persistently in the pol conformation by the presence of the helicase at the replication fork, the holoenzyme stimulates the helicase unwinding rate and prevents helicase slippage.
We, however, with the present data cannot comment on the exact number DNA base pairs destabilized or how it is achieved. The stimulation of both enzyme activities is mediated through the DNA and no specific strong protein—protein interaction is necessary allowing for homologous and heterologous coupling of holoenzymes and helicases to produce rapid and processive strand displacement activity so long as the polymerase primer extension rate is faster than the helicase unwinding rate.
Collaborative model for leading-strand DNA synthesis. A Diagram representing the coupling of the T4 holoenzyme and helicase in which the holoenzyme destabilizes the first few base pairs of the duplex DNA to enhance the helicase unwinding and the helicase prevents the base pair from reannealing thereby maintaining the holoenzyme in the polymerization conformation. The measured T4 helicase unwinding light blue symbols and T4 holoenzyme primer extension green symbols are shown for comparison.
C Comparisons between simulated grey lines and experimental red symbols results for the coupled replication rates V rep as a function of force left panel ; ATP concentration central panel and dNTP concentration right panel are shown.
Our collaborative model for coupled leading-strand DNA synthesis can be used to accurately reproduce our experimentally obtained results. Although not explicitly shown in Figure 5 A the polymerase is associated with the gp45 clamp protein that may dissociate in response to an applied force. According to our collaborative model, the coupled replication rate should be limited by the rate of the slowest enzyme under the experimental conditions.
The two main elements of the collaborative model are the prevention of the holoenzyme stalling by the presence of the helicase at the DNA fork and the holoenzyme induced destabilization of the DNA fork that stimulates helicase unwinding.
These two elements are most probably present in many replicative systems; on the one hand, replicative helicases are hexameric ring-like enzymes that encircle one strand of the DNA while stericly excluding the complementary strand; on the other hand, results obtained with different replicative holoenzymes show that, when stabilized in the pol active conformation, the polymerase is able to efficiently destabilize the DNA duplex.
Therefore, the collaborative model proposed here likely describes a general strategy in leading-strand coupling. This collaborative strategy may give the replisome the needed flexibility to coordinate leading- and lagging-strand synthesis and, at the same time, provide the tight coupling that prevents replisome disassembly.
The collaborative model between helicases and holoenzymes that we propose is not exclusive and does not preclude additional specific protein interactions or protein interaction networks to promote rapid DNA replication by replisomes.
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