Helicase Translocation Research Articles

Potent in vitro Interference of Fleroxacin in DNA-binding, Unwinding and ATPase Activities of Bloom Helicase

ObjectiveTo study the effect of fleroxacin (FLRX) on biological properties of Bloom (BLM) helicase catalytic core (BLM642-1290 helicase) in vitro and the molecular mechanism of interaction between the two molecules. MethodsDNA-binding and unwinding activities of BLM642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra. ResultsThe DNA unwinding and ATPase activities of BLM642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands. ConclusionFLRX may affect the biological activities and conformation of BLM642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.

Direct Observation of RecBCD Helicase as Single-Stranded DNA Translocases

The heterotrimeric Escherichia coli RecBCD enzyme comprises two helicase motors with different polarities: RecB (3'-to-5') and RecD (5'-to-3'). This superfamily I helicase is responsible for initiating DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (>200 nt) with no apparent secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt-end DNA, and translocate along the DNA substrates containing an ssDNA gap, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over a long gap in either 3'-to-5' or 5'-to-3' ssDNA direction indicates that RecBCD helicase possesses ssDNA translocase activities in both polarities. Most RecBCD active tethers showed full translocation across the ssDNA to the dsDNA region, with about 19% of enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. In addition, we prepared DNA substrates containing two opposite polarities (5'-to-3' and 3'-to-5') of ssDNA regions intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with less than 40% of tethers dissociating when entering into the second ssDNA region. These results suggest a model that RecBCD is able to switch between ssDNA translocases and rethread the other strand of ssDNA.

ATPase coupling in the processive RNA helicase NS3 from hepatitis C virus

Hepatitis C virus (HCV) NS3 motor protein contains both RNA helicase and serine protease activities that are vital to HCV replication in infected cells. NS3 belongs to the DExD/H‐box family of RNA helicases, which recognize short structured stretches of RNA and utilize ATP to remodel RNA and RNA‐protein complex structures. In contrast, NS3 is hypothesized to processively unwind the + and ‐ strands of the entire HCV RNA genome during viral replication, which likely requires that NS3 ATPase activity is tightly coupled to helicase translocation. To characterize the coupling of NS3 ATPase and translocation activities, we utilized equilibrium binding, steady‐state and transient kinetic biophysical methods. We compared a series of RNA substrates and found that RNA length affects both the concentration‐dependence and maximal rate of the RNA‐stimulated ATPase activity. Using transient kinetics, we demonstrated that the steady‐state rate of ATPase cycling is partially limited by a nucleotide‐dependent isomerization of the NS3‐RNA complex. On shorter RNAs, translocating NS3 protomers fall off the 5′ end of the nucleic acid track, and undergo this isomerization upon rebinding the ssRNA in order to restart productive translocation and rapid RNA‐stimulated ATPase. NS3 with an intact protease domain engages more productively with the RNA lattice, which increases apparent processivity of the ssRNA translocase.

Direct Observation of Recbcd Helicase as ssDNA Translocases

The E. coli RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB (3’-to-5’) and RecD (5’-to-3’). This Superfamily I helicase is responsible for the initiation of DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (> 200 nt) without secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt duplex DNA, and translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over long ssDNA gap in either 3’-to-5’ or 5’-to-3’ direction indicates that both RecB and RecD are ssDNA translocases. We also applied continuous force (∼ 0.2 pN) to stretch DNA substrates and to remove any potentially transient looped ssDNA structure, and observed continuous translocation of RecBCD. It confirms the ssDNA translocase activities of RecBCD helicase. About 78 % of active tethers showed full translocation across the ssDNA to the dsDNA region, and the other 22 % enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. We also prepared a double-gapped substrate containing two regions of ssDNA with opposite polarities (5’-to-3’ and 3’-to-5’) intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with 20 - 40 % of tethers dissociating while entering the second ssDNA region. These results suggest a mechanism that RecBCD is able to switch motors and rethread into the other strand after translocating along an ssDNA gapped region.

Coupling of Hexameric Helicase and DNA Polymerase in T7 Replisome during DNA Replication

Bacteriophage T7 replisome is one of the simplest replication machinery and we study the complexities of replication mechanism exploring this system. T7 gene 4 protein (gp4) assembles as a hexameric ring-shaped helicase with translocation polarity of 5' to 3' on ssDNA and duplex DNA unwinding during DNA replication. This protein also has a primase domain that generates short RNA primers for priming lagging strand DNA synthesis. It is known that T7 helicase coordinates with T7 DNA polymerase (T7gp5 -Thioredoxin of E. coli) in replisome to catalyze both leading and lagging strand DNA synthesis. However, functional and the physical extent of this relationship is not understood well. We have analyzed chemical coupling and physical proximity of these two motors during DNA replication in this study. Long standing question in the field has been that how does helicase couple the chemical energy of nucleotide hydrolysis for its translocation during DNA replication? This question could not be answered reliably so far due to the complexity and incompatibility of the NTP hydrolysis and helicase translocation activities. using pre-steady state kinetics approach combined with a novel coupling assay we have tackled this question. This is first ever report for the chemical step size determination for the helicase in replisome. Fine details of the energy coupling mechanism of the helicase in replisome are also revealed in these results. Second part of the study demonstrated the relative position of helicase and DNA polymerase in the replisome at the fork during DNA replication.

Structure-Based Simulations of the Translocation Mechanism of the Hepatitis C Virus NS3 Helicase along Single-Stranded Nucleic Acid

The NS3 helicase of Hepatitis C virus is an ATP-fueled molecular motor that can translocate along single-stranded (ss) nucleic acid, and unwind double-stranded nucleic acids. It makes a promising antiviral target and an important prototype system for helicase research. Despite recent progress, the detailed mechanism of NS3 helicase remains unknown. In this study, we have combined coarse-grained (CG) and atomistic simulations to probe the translocation mechanism of NS3 helicase along ssDNA. At the residue level of detail, our CG simulations have captured functionally important interdomain motions of NS3 helicase and reproduced single-base translocation of NS3 helicase along ssDNA in the 3′–5′ direction, which is in good agreement with experimental data and the inchworm model. By combining the CG simulations with residue-specific perturbations to protein-DNA interactions, we have identified a number of key residues important to the translocation machinery that agree with previous structural and mutational studies. Additionally, our atomistic simulations with targeted molecular dynamics have corroborated the findings of CG simulations and further revealed key protein-DNA hydrogen bonds that break/form during the transitions. This study offers, to our knowledge, the most detailed and realistic simulations of translocation mechanism of NS3 helicase. The simulation protocol established in this study will be useful for designing inhibitors that target the translocation machinery of NS3 helicase, and for simulations of a variety of nucleic-acid-based molecular motors.

PcrA-mediated disruption of RecA nucleoprotein filaments—essential role of the ATPase activity of RecA

The essential DNA helicase, PcrA, regulates recombination by displacing the recombinase RecA from the DNA. The nucleotide-bound state of RecA determines the stability of its nucleoprotein filaments. Using single-molecule fluorescence approaches, we demonstrate that RecA displacement by a translocating PcrA requires the ATPase activity of the recombinase. We also show that in a ‘head-on collision’ between a polymerizing RecA filament and a translocating PcrA, the RecA K72R ATPase mutant, but not wild-type RecA, arrests helicase translocation. Our findings demonstrate that translocation of PcrA is not sufficient to displace RecA from the DNA and assigns an essential role for the ATPase activity of RecA in helicase-mediated disruption of its filaments.

The inhibitory effects of vinylphosphonate-linked thymidine dimers on the unidirectional translocation of PcrA helicase along DNA: a molecular modelling study

The PcrA DNA helicases are important bacterial enzymes and quintessential examples of molecular motors. Through conformational changes caused by ATP hydrolysis, they move along the template double helix, breaking the hydrogen bonds holding the two strands together, and separating the template chains so that the genetic information can be accessed. The flexibility of the DNA backbone is essential for the unidirectional translocation of PcrA. A modified DNA substrate with reduced backbone rotational flexibility (via an incorporated vinylphosphonate linkage) has previously been designed and tested as a helicase substrate. The results show that a single modification on the backbone is sufficient to inhibit the activity of PcrA. In this paper a range of molecular simulation methods have been applied to examine the structural origins of this inhibitory effect, as it tests our theories of the mechanism of action of this motor. We observe that the chemical modification has different effects on the energetics of DNA translocation through the protein as it reaches different sub-sites.

Using a Single-Molecule Method to Visualize RecBCD Helicase Translocation along Single-Stranded DNA

The E. coli RecBCD helicase initiates the repair of double strand DNA break in the homologous recombination pathway. RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB is a 3’-to- 5’ helicase and RecD is a 5’-to-3’ helicase. How RecBCD unwinds and translocates along duplex DNA is not clearly defined. Here we used a single-molecule tethered particle motion (TPM) experiment to visualize the RecBCD helicase translocation over long distance single-stranded (ss) DNA. We first prepared DNA substrates containing a > 200 nt long, unstructured ssDNA gap flanked by double-stranded DNA for RecBCD loading. In the TPM experiments, the bead-labeled, biotinylated RecBCD helicases are found to recognize and bind to the blunt, double-stranded DNA end, and successfully translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion amplitude. Successful observation of RecBCD translocation over long ssDNA gap in either 3’-to- 5’ or 5’-to-3’ direction indicates that wild-type RecBCD functions an ssDNA translocase.

Promyelocytic leukemia protein interacts with werner syndrome helicase and regulates double-strand break repair in γ-irradiation-induced DNA damage responses

We show here that γ-irradiation leads to the translocation of endogenous Werner syndrome helicase (WRN) from nucleoli to nucleoplasmic DNA double strand breaks (DSBs), and WRN plays a role in damage repair. The relocation of WRN after irradiation was perturbed by promyelocytic leukemia protein (PML) knockdown and enhanced by PML IV overexpression. PML IV physically interacted with WRN after irradiation. Amino acids (a.a.) 394 to 433 of PML were necessary for this interaction and the nucleoplasmic translocation of WRN and were involved in DSB repair and cellular sensitivity to γ-irradiation. Taken together, our results provide molecular support for a model in which PML IV physically interacts with and regulates the translocation of WRN for DNA damage repair through its 394-433 a.a. domain.

The ATPase cycle of the RNA helicase protein NS3 from hepatitis C virus

The hepatitis C virus (HCV) NS3 protein possesses both RNA helicase and serine protease activities that are vital to HCV replication in infected cells. NS3 is also a more effective RNA helicase with the protease domain intact. NS3 is a member of the DExD/H‐box RNA helicase family, which recognize and remodel short structured stretches of RNA during every stage of RNA metabolism from synthesis to degradation. However, NS3 plays an important role in unwinding the + and − strands of the HCV genome during viral replication. This role requires ATPase activity that is tightly coupled with rapid helicase translocation and high processivity in concert with the viral RNA polymerase. To characterize the coupling of ATPase with helicase translocation activity of NS3, we utilized equilibrium binding and kinetic biophysical methods. By comparing a series of RNA substrates, we show that maximal RNA stimulation of ATPase activity occurs with very long RNAs, which affect both KM,RNA and kcat. The rate of ATPase cycling and helicase translocation along the RNA may be partially limited by rebinding NS3 to RNA following dissociation from the nucleic acid lattice during the ATPase cycle. The intact protease domain affords more productive engagement with the RNA lattice, which increases the ATPase‐translocase coupling efficiency (ATP consumed per RNA nt/bp travelled). An NSF CAREER Award (MCB‐0546353 to E.D.L.C.) supports this research.

Coupling Translocation with Nucleic Acid Unwinding by NS3 Helicase

NS3 helicase from hepatitis C virus (HCV) uses the free energy from ATP hydrolysis to translocate along single stranded DNA (ssDNA) or RNA, unwind double-stranded (ds) duplex, or displace proteins along the track. In this work, we present a semi-quantitative model for translocation and unwinding activities of monomeric NS3 helicase. The model is based on structural, biochemical and single molecule measurements of the helicase. First, we have assumed that NS3 helicase walks unidirectionally by alternately moving its two translocase domains forward one nucleotide (nt) at a time, as that being suggested for a similar helicase PcrA. Second, in our model NS3 helicase displays diffusive character upon ATP binding (before helicase dissociation), due to weak affinity of the helicase to the ssDNA. The helicase diffusion leads to occasional backward steps and increases displacement fluctuations that are supposedly detectable from single molecule experiments. Further, our model suggests that NS3 helicase interacts with the duplex at the ss-ds junction such that (i) the helicase can actively unwind the duplex by reducing free energy of base pairing /stacking at the duplex end; (ii) the helicase can be stabilized at the junction by its favorable association with the duplex backbone. By fitting with NS3 unwinding data from single molecule optical-tweezer measurements, our model explains sequence dependences of the unwinding velocity, efficiency, and helicase dissociation rate. In particular, based on the experimental data, we have quantified how active the NS3 helicase is in its unwinding, and estimated how fast the helicase diffuses in its ATP bound state. The generic features of coupling ssDNA/RNA translocation with duplex unwinding presented in our NS3 helicase model may apply as well to other similar systems.

Short Double-stranded RNAs with an Overhanging 5′ ppp-Nucleotide, as Found in Arenavirus Genomes, Act as RIG-I Decoys

Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended (5' ppp)dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended (5' ppp)dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5' ppp. In contrast, (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5' ppp group.

Inhibition of RNA Helicases of ssRNA(+) Virus Belonging to Flaviviridae, Coronaviridae and Picornaviridae Families.

Many viral pathogens encode the motor proteins named RNA helicases which display various functions in genome replication. General strategies to design specific and selective drugs targeting helicase for the treatment of viral infections could act via one or more of the following mechanisms: inhibition of the NTPase activity, by interferences with ATP binding and therefore by limiting the energy required for the unwinding and translocation, or by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state; inhibition of nucleic acids binding to the helicase; inhibition of coupling of ATP hydrolysis to unwinding; inhibition of unwinding by sterically blocking helicase translocation. Recently, by in vitro screening studies, it has been reported that several benzotriazole, imidazole, imidazodiazepine, phenothiazine, quinoline, anthracycline, triphenylmethane, tropolone, pyrrole, acridone, small peptide, and Bananin derivatives are endowed with helicase inhibition of pathogen viruses belonging to Flaviviridae, Coronaviridae, and Picornaviridae families.

Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3'-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.

Multiple Escherichia coli RecQ Helicase Monomers Cooperate to Unwind Long DNA Substrates: A FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY STUDY

The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3'-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37 degrees C, whereas only modest or no cooperativity was observed at 25 degrees C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37 degrees C but not at 25 degrees C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.

Ensemble and single-molecule fluorescence-based assays to monitor DNA binding, translocation, and unwinding by iron–sulfur cluster containing helicases

Many quantitative approaches for analysis of helicase–nucleic acid interactions require a robust and specific signal, which reports on the presence of the helicase and its position on a nucleic acid lattice. Since 2006, iron–sulfur (FeS) clusters have been found in a number of helicases. They serve as endogenous quenchers of Cy3 and Cy5 fluorescence which can be exploited to characterize FeS cluster containing helicases both in ensemble-based assays and at the single-molecule level. Synthetic oligonucleotides site-specifically labeled with either Cy3 or Cy5 can be used to create a variety of DNA substrates that can be used to characterized DNA binding, as well as helicase translocation and unwinding. Equilibrium binding affinities for ssDNA, duplex and branched DNA substrates can be determined using bulk assays. Identification of preferred cognate substrates, and the orientation and position of the helicase when bound to DNA can also be determined by taking advantage of the intrinsic quencher in the helicase. At the single-molecule level, real-time observation of the helicase translocating along DNA either towards the dye or away from the dye can be used to determine the rate of translocation by the helicase on ssDNA and its orientation when bound to DNA. The use of duplex substrates can reveal the rate of unwinding and processivity of the helicase. Finally, the FeS cluster can be used to visualize protein–protein interactions, and to examine the interplay between helicases and other DNA binding proteins on the same DNA substrate.

Reeling in DNA One Base at A Time: pcrA Translocation Coupled to DNA Looping Dismantles RecA Filaments

The mechanism of helicase translocation on DNA remains controversial and the translocase activity driving their non-canonical functions such as protein displacement is poorly understood. Here, we used single molecule fluorescence assays to study a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, and discovered a progressive looping of ssDNA that is tightly coupled to PcrA translocation on DNA. Variance analysis of hundreds of looping events by a single protein demonstrated that PcrA translocates on ssDNA in uniform steps of 1 nt, reconciling discrepancies in previous structural and biochemical studies. On the forked DNA, rather than acting on the leading strand to unwind the duplex, PcrA anchored itself to the duplex junction and reeled in the lagging strand using its 3′-5′ translocation activity. PcrA maintained the open conformation, not the closed conformation observed in crystallographic analysis, during looping-coupled translcation. This activity could rapidly dismantle a preformed RecA filament even at 1nM PcrA, suggesting that the translocation activity and structure-specific DNA binding are responsible for removal of potentially deleterious recombination intermediates.

An in Trans Interaction at the Interface of the Helicase and Primase Domains of the Hexameric Gene 4 Protein of Bacteriophage T7 Modulates Their Activities

DNA helicase and primase are essential for DNA replication. The helicase unwinds the DNA to provide single-stranded templates for DNA polymerase. The primase catalyzes the synthesis of oligoribonucleotides for the initiation of lagging strand synthesis. The two activities reside in a single polypeptide encoded by gene 4 of bacteriophage T7. Their coexistence within the same polypeptide facilitates their coordination during DNA replication. One surface of helix E within the helicase domain is positioned to interact with the primase domain and the linker connecting the two domains within the functional hexamer. The interaction occurs in trans such that helix E interacts with the primase domain and the linker of the adjacent subunit. Most alterations of residues on the surface of helix E (Arg(404), Lys(408), Tyr(411), and Gly(415)) eliminate the ability of the altered proteins to complement growth of T7 phage lacking gene 4. Both Tyr(411) and Gly(415) are important in oligomerization of the protein. Alterations G415V and K408A simultaneously influence helicase and primase activities in opposite manners that mimic events observed during coordinated DNA synthesis. The results suggest that Asp(263) located in the linker of one subunit can interact with Tyr(411), Lys(408), or Arg(404) in helix E of the adjacent subunit depending on the oligomerization state. Thus the switch in contacts between Asp(263) and its three interacting residues in helix E of the adjacent subunit results in conformational changes that modulate helicase and primase activity.

Fuel Specificity of the Hepatitis C Virus NS3 Helicase

The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of an oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in solution, such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTP. 3′-Deoxy-NTPs generally support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of an intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson–Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2′ and 3′ carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn 2+ supporting about 10 times faster unwinding than Mg 2+. Unlike Mg 2+, Mn 2+ does not support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction.