Coupling translocation with nucleic acid unwinding by NS3 helicase

We present a semiquantitative model for translocation and unwinding activities of monomeric nonstructural protein 3 (NS3) helicase. The model is based on structural, biochemical, and single-molecule measurements. The model predicts that the NS3 helicase actively unwinds duplex by reducing more than 50% the free energy that stabilizes base pairing/stacking. The unwinding activity slows the movement of the helicase in a sequence-dependent manner, lowering the average unwinding efficiency to less than 1 bp per ATP cycle. When bound with ATP, the NS3 helicase can display significant translocational diffusion. This increases displacement fluctuations of the helicase, decreases the average unwinding efficiency, and enhances the sequence dependence. Also, interactions between the helicase and the duplex stabilize the helicase at the junction, facilitating the helicase's unwinding activity while preventing it from dissociating. In the presence of translocational diffusion during active unwinding, the dissociation rate of the helicase also exhibits sequence dependence. Based on unwinding velocity fluctuations measured from single-molecule experiments, we estimate the diffusion rate to be on the order of 10 s^− 1 . The generic features of coupling single-stranded nucleic acid translocation with duplex unwinding presented in this work may apply generally to a class of helicases.     

Archaeal Hel308 domain V couples DNA binding to ATP hydrolysis and positions DNA for unwinding over the helicase ratchet

Hel308 and PolQ are paralogues with roles promoting genome stability in archaea and higher eukaryotes. The context in which they act is not clear, although Hel308 helicase from archaea may interact with abnormal replication forks. The atomic structure of archaeal Hel308 from Archaeoglobus fulgidus in complex with DNA was recently reported and has given insights into the mechanisms of superfamily-2 helicases generally. An intriguing aspect of the structure was the positioning of a C-terminal domain V relative to single-stranded DNA and to the helicase ratchet domain IV. We have mutagenised a triplet of arginine residues in domain V of archaeal Hel308 to assess the effects on DNA binding, unwinding, and ATPase activities. Our observations can now be interpreted in light of the atomic structure. We describe crucial roles for domain V as a brake on ATP hydrolysis by coupling it to binding single-stranded DNA and in positioning DNA relative to the helicase ratchet domain IV for efficient unwinding of forked DNA.     

Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe(523), positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe(523) with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe(523) is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe(523) may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe(523) moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.     

DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies

The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding.     

Grip it and rip it: Structural mechanisms of DNA helicase substrate binding and unwinding

Maintenance and faithful transmission of genomic information depends on the efficient execution of numerous DNA replication, recombination, and repair pathways. Many of the enzymes that catalyze steps within these pathways require access to sequence information that is buried in the interior of the DNA double helix, which makes DNA unwinding an essential cellular reaction. The unwinding process is mediated by specialized molecular motors called DNA helicases that couple the chemical energy derived from nucleoside triphosphate hydrolysis to the otherwise non‐spontaneous unwinding reaction. An impressive number of high‐resolution helicase structures are now available that, together with equally important mechanistic studies, have begun to define the features that allow this class of enzymes to function as molecular motors. In this review, we explore the structural features within DNA helicases that are used to bind and unwind DNA. We focus in particular on “aromatic‐rich loops” that allow some helicases to couple single‐stranded DNA binding to ATP hydrolysis and “wedge/pin” elements that provide mechanical tools for DNA strand separation when connected to translocating motor domains.     

Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase

Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased approximately 10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.     

BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation

Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3′–5′ DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single‐molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can ‘measure’ how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild‐type BLM and hRPA was necessary for unwinding reinitiation on hRPA‐coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates.     

Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: the effect of the 3' arm and the stability of the dsDNA on the unwinding activity of the Escherichia coli DnaB helicase

The effect of two structural elements of a replication DNA fork substrate, the length of the 3' arm of the fork and the stability of the double-stranded DNA (dsDNA) part, on the kinetics of the dsDNA unwinding by the Escherichia coli hexameric helicase DnaB protein has been examined under single turnover conditions using the rapid quench-flow technique. The length of the 3' arm of the replication fork, i.e. the number of nucleotides in the arm, is a major structural factor that controls the unwinding rate and processivity of the helicase. The data show the existence of an optimal length of the 3' arm where there is the highest unwinding rate and processivity, indicating that during the unwinding process, the helicase transiently interacts with the 3' arm at a specific distance on the arm with respect to the duplex part of the DNA. Moreover, the area on the enzyme that engages in interactions has also a discrete size. For DNA substrates with the 3' arm containing 14, or less, nucleotide residues, the DnaB helicase becomes a completely distributive enzyme. However, the 3' arm is not a "specific activating cofactor" in the unwinding reaction. Rather, the 3' arm plays a role as a mechanical fulcrum for the enzyme, necessary to provide support for the advancing large helicase molecule on the opposite strand of the DNA. Binding of ATP is necessary to engage the 3' arm with the DnaB helicase, but it does not change the initial distribution of complexes of the enzyme with the DNA fork substrate. Stability of the dsDNA has a significant effect on the unwinding rate and processivity. The unwinding rate constant is a decreasing linear function of the fractional content of GC base-pairs in the dsDNA, indicating that the activation of the unwinding step is proportional to the stability of the nucleic acid.     

Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method

Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining approximately 11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(+/- 0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25 degrees C unwinds approximately 291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P = 0.89(+/- 0.03), little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.     

Fluorescence stopped-flow studies of single turnover kinetics of E.coli RecBCD helicase-catalyzed DNA unwinding

We have developed and optimized a stopped-flow fluorescence assay for use in studying DNA unwinding catalyzed by Escherichia coli RecBCD helicase. This assay monitors changes in fluorescence resonance energy transfer (FRET) between a pair of fluorescent probes (Cy3 donor and Cy5 acceptor) placed on opposite sides of a nick in duplex DNA. As such, this is an "all-or-none" DNA unwinding assay. Single turnover DNA unwinding experiments were performed using a series of eight fluorescent DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. The time-courses obtained by monitoring Cy3 fluorescence display a distinct lag phase that increases with increasing duplex DNA length, reflecting the transient formation of partially unwound DNA intermediates. These Cy3 FRET time-courses are identical with those obtained using a chemical quenched-flow kinetic assay developed previously. The signal from the Cy5 fluorescence probe shows additional effects that appear to specifically monitor the RecD helicase subunit. The continuous nature of this fluorescence assay enabled us to acquire more precise time-courses for many more duplex DNA lengths in a significantly reduced amount of time, compared to quenched-flow methods. Global analysis of the Cy3 and Cy5 FRET time-courses, using an n-step sequential DNA unwinding model, indicates that RecBCD unwinds duplex DNA with an average unwinding rate constant of kU = 200(+/-40) steps s(-1) (mkU = 680(+/-12)bp s(-1)) and an average kinetic step size, m = 3.4 (+/-0.6) bp step(-1) (5 mM ATP, 10 mM MgCl(2), 30 mM NaCl, pH 7.0, 5% (v/v) glycerol, 25.0 degrees C), in excellent agreement with the kinetic parameters determined using quenched-flow techniques. Under these same conditions, the RecBC enzyme unwinds DNA with a very similar rate. These methods will facilitate detailed studies of the mechanisms of DNA unwinding and translocation of the RecBCD and RecBC helicases.     

Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents

Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.     

T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA

DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.     

Effects of temperature and ATP on the kinetic mechanism and kinetic step-size for E.coli RecBCD helicase-catalyzed DNA unwinding

The kinetic mechanism by which Escherichia coli RecBCD helicase unwinds duplex DNA was studied using a fluorescence stopped-flow method. Single turnover DNA unwinding experiments were performed using a series of fluorescently labeled DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. All or no DNA unwinding time courses were obtained by monitoring the changes in fluorescence resonance energy transfer between a Cy3 donor and Cy5 acceptor fluorescent pair placed on opposite sides of a nick in the duplex DNA. From these experiments one can determine the average rates of DNA unwinding as well as a kinetic step-size, defined as the average number of base-pairs unwound between two successive rate-limiting steps repeated during DNA unwinding. In order to probe how the kinetic step-size might relate to a mechanical step-size, we performed single turnover experiments as a function of [ATP] and temperature. The apparent unwinding rate constant, kUapp, decreases with decreasing [ATP], exhibiting a hyperbolic dependence on [ATP] (K1/2=176(+/-30) microM) and a maximum rate of kUapp=204(+/-4) steps s(-1) (mkUapp=709(+/-14) bp s(-1)) (10 mM MgCl2, 30 mM NaCl (pH 7.0), 5% (v/v) glycerol, 25.0 degrees C). kUapp also increases with increasing temperature (10-25 degrees C), with Ea=19(+/-1) kcal mol(-1). However, the average kinetic step-size, m=3.9(+/-0.5) bp step(-1), remains independent of [ATP] and temperature. This indicates that even though the values of the rate constants change, the same elementary kinetic step in the unwinding cycle remains rate-limiting over this range of conditions and this kinetic step remains coupled to ATP binding. The implications of the constancy of the measured kinetic step-size for the mechanism of RecBCD-catalyzed DNA unwinding are discussed.     

Impediment of E. coli UvrD by DNA‐destabilizing force reveals a strained‐inchworm mechanism of DNA unwinding

Escherichia coli UvrD is a non‐ring‐shaped model helicase, displaying a 3′—5′ polarity in DNA unwinding. Using a transverse magnetic tweezer and DNA hairpins, we measured the unwinding kinetics of UvrD at various DNA‐destabilizing forces. The multiform patterns of unwinding bursts and the distributions of the off‐times favour the mechanism that UvrD unwinds DNA as a dimer. The two subunits of the dimer coordinate to unwind DNA processively. They can jointly switch strands and translocate backwards on the other strand to allow slow (～40 bp/s) rewinding, or unbind simultaneously to allow quick rehybridization. Partial dissociation of the dimer results in pauses in the middle of the unwinding or increases the translocation rate from ～40 to ～150 nt/s in the middle of the rewinding. Moreover, the unwinding rate was surprisingly found to decrease from ～45 to ～10 bp/s when the force is increased from 2 to 12 pN. The results lead to a strained‐inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer.     

The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3′-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5′-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.     

Mechanochemistry of t7 DNA helicase

The bacteriophage T7 helicase is a ring-shaped hexameric motor protein that unwinds double-stranded DNA during DNA replication and recombination. To accomplish this it couples energy from the nucleotide hydrolysis cycle to translocate along one of the DNA strands. Here, we combine computational biology with new biochemical measurements to infer the following properties of the T7 helicase: (1) all hexameric subunits are catalytic; (2) the mechanical movement along the DNA strand is driven by the binding transition of nucleotide into the catalytic site; (3) hydrolysis is coordinated between adjacent subunits that bind DNA; (4) the hydrolysis step changes the affinity of a subunit for DNA allowing passage of DNA from one subunit to the next. We construct a numerical optimization scheme to analyze transient and steady-state biochemical measurements to determine the rate constants for the hydrolysis cycle and determine the flux distribution through the reaction network. We find that, under physiological and experimental conditions, there is no dominant pathway; rather there is a distribution of pathways that varies with the ambient conditions. Our analysis methods provide a systematic procedure to study kinetic pathways of multi-subunit, multi-state cooperative enzymes.     

Partial purification and characterization of a DNA helicase from chloroplasts of Glycine max

A DNA helicase activity was detected in extracts of purified chloroplasts from the SB-1 cell line of Glycine max and partially purified by column chromatography on DEAE cellulose, phosphocellulose, and single-stranded DNA cellulose. The chloroplast helicase has a DNA-dependent ATPase activity, and its strand displacement activity is strictly dependent upon the presence of a nucleoside triphosphate and Mg²⁺ or Mn²⁺. Strand displacement activity does not require a free unannealed single-strand or replication fork-like structure.