A General Model for Nucleic Acid Helicases and Their “Coupling” within Macromolecular Machines

Hippel, Peter H. von; Delagoutte, Emmanuelle

doi:10.1016/s0092-8674(01)00203-3

Cited by 182 publications

(182 citation statements)

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“…The 9-fold difference in ssDNA translocation and unwinding rates in Fig. 1 A and B) terms of a single base pair unwound or a single base translocated (k unwinding ͞k ssDNA transloation ) corresponds to a free-energy difference of 1.2 kcal͞mol, a value close to the average free energy of opening a single base pair of DNA (2,13). These results imply that T7 helicase is primarily a ssDNA translocase and the duplex DNA poses a barrier to its movement along ssDNA, although we cannot rule out the possibility that the physical presence of the large helicase hexamer at the fork junction might perturb the duplex region at the junction.…”

Section: Discussionmentioning

confidence: 89%

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The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

Jeong

Levin

Patel

2004

Proc. Natl. Acad. Sci. U.S.A.

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Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on singlestranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp͞s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.H elicases are ubiquitous proteins that are involved in various DNA and RNA metabolic processes that require the separation of double-stranded (ds)DNA into single strands, the removal of secondary structures in RNA, or the dissociation of proteins from nucleic acids (1-4). To perform these functions, helicases use the chemical energy from NTP hydrolysis to drive the mechanical processes of translocation and nucleic acid strand separation. In this paper, we study the mechanism of DNA unwinding by bacteriophage T7 helicase that is involved in DNA replication.During replication, the helicase has to unwind a long stretch of DNA, and that requires the helicase to couple strandseparation activity to translocation. The mechanisms of these critical processes of the helicase reaction are largely unknown. It is becoming evident that helicases can move unidirectionally along nucleic acid and displace bonded moieties along their path without specifically interacting with these moieties (5-8). Thus, unidirectional translocation is a basic activity that helicases can perform without requiring interactions with the duplex DNA. Nucleic acid strand separation is a thermodynamically unfavorable process and it is made feasible by the binding of the helicase to the newly unwound strands. Numerous mechanisms of unwinding have been proposed (2-4, 9-13), but additional experimental data are needed to distinguish between these mechanisms. For the monomeric or dimeric helicases such as the Escherichia coli PcrA and Rep helicases (14-16), it has been proposed that unwinding occurs by an active mechani...

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Section: Discussionmentioning

confidence: 89%

“…T7 helicase and most helicases make the unwinding process thermodynamically feasible by binding to the ssDNA (4,13). In addition to ssDNA binding, it has been proposed that helicases such as Rep and PcrA bind directly to the duplex DNA to destabilize it (14)(15)(16).…”

Section: Discussionmentioning

confidence: 99%

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

Jeong

Levin

Patel

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Numerous nucleic acid translocases were shown to move at rates limited by the thermal fraying of nucleic acid base pairs (7,28). In contrast to these enzymes, NS3 moves on nucleic acids faster than the rate imposed by the thermal fraying of base pairs.…”

Section: Discussionmentioning

confidence: 99%

“…Despite recent progress in the study of these motor proteins (2-4), the physical mechanisms by which they move and catalyze the strand separation are not well understood. Helicase models ranging from a pure Brownian ratchet to a pure power stroke action have been discussed (5)(6)(7)(8), but experimental data to support them for RNA helicases have been lacking. The hepatitis C virus NS3 protein is a superfamily 2, 3Ј to 5Ј RNA helicase (9) known to be essential for virus replication and, thus, an antiviral drug target (10).…”

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confidence: 99%

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

Cheng¹,

Dumont

Tinoco³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

149

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RNA helicases regulate virtually all RNA-dependent cellular processes. Although much is known about helicase structures, very little is known about how they deal with barriers in RNA and the factors that affect their processivity. The hepatitis C virus encodes NS3, an RNA helicase that is essential for viral RNA replication. We have used optical tweezers to determine at the single-molecule level how the local stability of the RNA substrate affects the enzyme rate of strand separation, whether separation occurs by an active or a passive mechanism, and whether processivity is affected. We show that sequence barriers in RNA modulate NS3 activity. NS3 processivity depends on barriers ahead of the opening fork. Our results rule out a model where NS3 passively waits for the thermal fraying of double-stranded RNA. Instead, we find that NS3 destabilizes the duplex before separating the strands. Failure to do so before a strong barrier leads to helicase dissociation and limits the processivity of the enzyme.hairpin ͉ molecular motors ͉ optical tweezers ͉ processivity A wide range of RNA metabolic activities (1) require the breaking of base pairs in double-stranded RNA (dsRNA) by RNA helicases. Despite recent progress in the study of these motor proteins (2-4), the physical mechanisms by which they move and catalyze the strand separation are not well understood. Helicase models ranging from a pure Brownian ratchet to a pure power stroke action have been discussed (5-8), but experimental data to support them for RNA helicases have been lacking. The hepatitis C virus NS3 protein is a superfamily 2, 3Ј to 5Ј RNA helicase (9) known to be essential for virus replication and, thus, an antiviral drug target (10). Recently, NS3 has been the subject of single-molecule manipulation studies (4), which revealed that NS3 makes steps of 11 bp each made up of three substeps (4). RNA molecules contain various kinetic barriers to mechanical unfolding (11). How these barriers influence the activity of motor proteins that work on RNA will depend on the mechanism of the motor. To probe the molecular mechanism of NS3 unwinding activity, we have designed and characterized RNA molecules with various mechanical unfolding barriers and used single-molecule methods to investigate how these barriers affect the velocity, pausing, and processivity of the helicase in real time. ResultsDesign and Characterization of RNA Hairpin Substrates. We first designed two RNA hairpin substrates that terminate both on a tetraloop (Fig. 1A) to monitor the response of a single NS3 helicase to weak and strong sequence barriers. Substrate RNA-AG has 30 A⅐U pairs followed by 30 G⅐C pairs; RNA-GA has the A⅐U and G⅐C sequences interchanged. The sequences within A⅐U and G⅐C regions were chosen to minimize the formation of alternative secondary structures other than the desired 60-bp hairpin. In our experiment, a single RNA hairpin was attached between a microsphere in an optical trap and a microsphere placed on the end of a micropipette through hybrid RNA-DNA handles to se...

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“…An important review of helicase mechanism by von Hippel and Delagoutte [7] synthesizes the thermodynamic and kinetic properties of helicases into a general framework and emphasizes the importance of considering helicase-NA binding, translocation, and strand-separation independently. Doering et al [8] developed a "flashing field" model specific to hexameric ring helicases.…”

Section: Introductionmentioning

confidence: 99%

A Motor that Makes Its Own Track: Helicase Unwinding of DNA

Betterton

Jülicher

2003

Phys. Rev. Lett.

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Helicase opening of double-stranded nucleic acids may be "active" (the helicase directly destabilizes the dsNA to promote opening) or "passive" (the helicase binds ssNA available due to a thermal fluctuation which opens part of the dsNA). We describe helicase opening of dsNA, based on helicases which bind single NA strands and move towards the double-stranded region, using a discrete "hopping" model. The interaction between the helicase and the junction where the double strand opens is characterized by an interaction potential. The form of the potential determines whether the opening is active or passive. We calculate the rate of passive opening for the helicase PcrA, and show that the rate increases when the opening is active. Finally, we examine how to choose the interaction potential to optimize the rate of strand separation. One important result is our finding that active opening can increase the unwinding rate by 7 fold compared to passive opening.

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A General Model for Nucleic Acid Helicases and Their “Coupling” within Macromolecular Machines

Abstract: in Table 1; most of these helicase functions are clearly required in other organisms as well. Helicase reactions tend to be driven by the hydrolysis of ATP or other nucleoside triphosphates (NTPs). This process is generally catalyzed by a cryptic ATPase activity carried by

Cited by 182 publications

References 57 publications

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

A Motor that Makes Its Own Track: Helicase Unwinding of DNA

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