How directional translocation is regulated in a DNA helicase motor

PcrA helicase from Bacillus stearothermophilus is one of the smallest motor proteins structurally known in full atomic detail. It translocates progressively from the 3' end to the 5' end of single-stranded DNA utilizing the free energy from ATP hydrolysis. The similarities in structure and reaction pathway between PcrA helicase and F1-ATPase suggest a similar mechanochemical mechanism at work in both systems. Previous studies of PcrA translocation demonstrated a domain stepping mechanism in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of two translocation domains is synchronized with alternating mobilities of the individual domains such that PcrA moves unidirectionally along single-stranded DNA. To substantiate this translocation mechanism, this study applies molecular dynamics simulations, elastic network theory, and multiple sequence alignment to analyze the system. The analysis provides further evidence that directional translocation of PcrA is regulated allosterically through synchronization of ATP hydrolysis and domain mobilities. We identify a set of essential residues coevolutionarily coupled in related helicases that should be involved in the allosteric regulation of these motor proteins.     

Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism

DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3'-tailed DNA duplex reveal a contact region that covers a significant region of the substrate both in the presence and absence of a non-hydrolysable analogue of ATP, ADPNP. However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single-stranded DNA translocation activities intact. These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support an 'active' mechanism for PcrA that involves two distinct ATP-dependent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.     

A nonuniform stepping mechanism for E. coli UvrD monomer translocation along single-stranded DNA

E. coli UvrD is an SF1 helicase involved in several DNA metabolic processes. Although a UvrD dimer is needed for helicase activity, a monomer can translocate with 3' to 5' directionality along single-stranded DNA, and this ATP-dependent translocation is likely involved in RecA displacement. In order to understand how the monomeric translocase functions, we have combined fluorescence stopped-flow kinetic methods with recently developed analysis methods to determine the kinetic mechanism, including ATP coupling stoichiometry, for UvrD monomer translocation along ssDNA. Our results suggest that the macroscopic rate of UvrD monomer translocation is not limited by each ATPase cycle but rather by a slow step (pause) in each translocation cycle that occurs after four to five rapid 1 nt translocation steps, with each rapid step coupled to hydrolysis of one ATP. These results suggest a nonuniform stepping mechanism that differs from either a Brownian motor or previous structure-based inchworm mechanisms.     

Differential ATP requirements distinguish the DNA translocation and DNA unwinding activities of the Escherichia coli PRI A protein

The Escherichia coli primosome is a mobile multiprotein DNA replication-priming apparatus that assembles at a specific site (termed a primosome assembly site (PAS] on single-stranded DNA-binding protein-coated single-stranded DNA. The PRI A protein (factor Y, protein n') is a PAS sequence-specific (d)ATPase as well as a DNA helicase and is believed to direct the assembly of the primosome at a PAS. In this report, the PRI A DNA helicase reaction is dissected in vitro, by use of a strand displacement assay, into three steps with distinct ATP requirements. First, the PRI A protein gains entry to the DNA via an ATP-independent, PAS sequence-specific binding event. Second, the PRI A protein translocates along the single-stranded DNA in the 3'----5' direction at a maximal rate of 90 nucleotides/s. DNA translocation requires ATP hydrolysis. The ATP concentration required to support half of the maximal translocation rate is 100 microM, which is identical to the Km for ATP of the PRI A protein DNA-dependent ATPase activity. Finally, the PRI A protein unwinds duplex DNA. The ATP concentration required for duplex DNA unwinding depends upon the length of the duplex region to be unwound. Displacement of a 24-nucleotide long oligomer required no more ATP than that required for the translocation of PRI A protein along single-stranded DNA, whereas displacement of a 390-nucleotide long DNA fragment required a 10-fold higher concentration of ATP than that required for oligomer displacement.     

PcrA helicase, a prototype ATP-driven molecular motor

Despite extensive studies, the mechanisms underlying molecular motor function are still poorly understood. Key to the mechanisms is the coupling of ATP hydrolysis to conformational changes of the motor protein. To investigate this coupling, we have conducted combined quantum mechanical/molecular mechanical simulations of PcrA helicase, a strikingly simple motor that translocates unidirectionally along single-stranded DNA (ssDNA). Our results reveal a close similarity in catalytic site structure and reaction pathway to those of F1-ATPase, and these similarities include a proton relay mechanism important for efficient ATP hydrolysis and an "arginine finger" residue that is key to the coupling of the chemical reaction to protein conformational changes. By means of in silico mutation studies, we identified the residue Q254 as being crucial for the coupling of ssDNA translocation to the actual catalytic event. Based on the present result for PcrA helicase and previous findings for F1-ATPase, we propose a general mechanism of ATP-driven molecular motor function.     

The study of interactions between DNA and PcrA DNA helicase by using targeted molecular dynamic simulations

DNA helicases are important enzymes involved in all aspects of nucleic acid metabolism, ranging from DNA replication and repair to recombination, rescue of stalled replication and translation. DNA helicases are molecular motors. Through conformational changes caused by ATP hydrolysis and binding, they move along the template double helix, break the hydrogen bonds between the two strands and separate the template chains, so that the genetic information can be accessed. In this paper, targeted molecular dynamic simulations were performed to study the important interactions between DNA and PcrA DNA helicase, which can not be observed from the crystal structures. The key residues on PcrA DNA helicase that have strong interactions with both double stranded DNA (ds-DNA) and single stranded DNA (ss-DNA) have been identified, and it was found that such interactions mostly exist between the protein and DNA backbone, which indicates that the translocation of PcrA is independent of the DNA sequence. The simulations indicate that the ds-DNA is separated upon ATP rebinding, rather than ATP hydrolysis, which suggests that the two strokes in the mechanism have two different major roles. Firstly, in the power stroke (ATP hydrolysis), most of the translocations of the bases from one pocket to the next occur. In the relaxation stroke (ATP binding), most of the ‘work’ is being done to ‘melt’ the DNA at the separation fork. Therefore, we propose a mechanism whereby the translocation of the ss-DNA is powered by ATP hydrolysis and the separation of the ds-DNA is powered by ATP binding.     

Single-stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis

Escherichia coli UvrD is an SF1A (superfamily 1 type A) helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded DNA (ssDNA) translocase but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500?μM), we proposed a nonuniform stepping mechanism in which a UvrD monomer translocates with biased (3' to 5') directionality while hydrolyzing 1 ATP per DNA base translocated, but with a kinetic step size of 4-5?nt/step, suggesting that a pause occurs every 4-5?nt translocated. To further test this mechanism, we examined UvrD translocation over a range of lower ATP concentrations (10-500?μM ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of ～1 ATP/DNA base translocated even at the lowest ATP concentration examined (10?μM), indicating that ATP hydrolysis is tightly coupled to forward translocation of a UvrD monomer along ssDNA with little slippage or futile ATP hydrolysis during translocation. The translocation kinetic step size remains constant at 4-5?nt/step down to 50?μM ATP but increases to ～7?nt/step at 10?μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP but with little futile ATP hydrolysis.     

A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase

Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid-binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid. Now we identify the stage of the ATPase cycle responsible for translocation and unwinding. We show that a rapid directional movement occurs upon helicase binding to DNA in the absence of ATP, resulting in opening of several base pairs. We propose that HCV helicase translocates as a Brownian motor with a simple two-stroke cycle. The directional movement step is fueled by single-stranded DNA binding energy while ATP binding allows for a brief period of random movement that prepares the helicase for the next cycle.     

Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine.

Use of the fluorescent base analogue 2-aminopurine has provided a direct demonstration of the translocation of PcrA helicase toward the 5'-end of single-stranded DNA. Single 2-aminopurine bases are introduced into otherwise standard oligonucleotides and produce a fluorescence signal when PcrA reaches their position. We demonstrate that random binding of PcrA to ssDNA is followed by translocation in an ATP-dependent manner toward the 5'-terminus at 80 bases per second at 20 degrees C. The data also provide information on the kinetics of ssDNA binding to the helicase and of the protein dissociation from the 5'-end of ssDNA. A full kinetic model is presented for ATP-dependent DNA translocation by PcrA helicase.     

Enzymatic mechanism of RNA translocation in double-stranded RNA bacteriophages

Many complex viruses acquire their genome by active packaging into a viral precursor particle called a procapsid. Packaging is performed by a viral portal complex, which couples ATP hydrolysis to translocation of nucleic acid into the procapsid. The packaging process has been studied for a variety of viruses, but the mechanism of the associated ATPase remains elusive. In this study, the mechanism of RNA translocation in double-stranded RNA bacteriophages is characterized using rapid kinetic analyses. The portal complex of bacteriophage 8 is a hexamer of protein P4, which exhibits nucleotide triphosphatase activity. The kinetics of ATP binding reveals a two-step process: an initial, fast, second-order association, followed by a slower, first-order phase. The slower phase exhibits a high activation energy and has been assigned to a conformational change. ATP binding becomes cooperative in the presence of RNA. Steady-state kinetics of ATP hydrolysis, which proceeds only in the presence of RNA, also exhibits cooperativity. On the other hand, ADP release is fast and RNA-independent. The steady-state rate of hydrolysis increases with the length of the RNA substrate indicating processive translocation. Raman spectroscopy reveals that RNA binds to P4 via the phosphate backbone. The ATP-induced conformational change affects the backbone of the bound RNA but leaves the protein secondary structure unchanged. This is consistent with a model in which cooperativity is induced by an RNA link between subunits of the hexamers and translocation is effected by an axial movement of the subunits relative to one another upon ATP binding.     

The ATPase Cycle of PcrA Helicase and Its Coupling to Translocation on DNA

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2′(3′)-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP·P_i being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P_i release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se.     

Mechanism of RecQ helicase mechanoenzymatic coupling reveals that the DNA interactions of the ADP-bound enzyme control translocation run terminations

The processing of various DNA structures by RecQ helicases is crucial for genome maintenance in both bacteria and eukaryotes. RecQ helicases perform active destabilization of DNA duplexes, based on tight coupling of their ATPase activity to moderately processive translocation along DNA strands. Here, we determined the ATPase kinetic mechanism of E. coli RecQ helicase to reveal how mechanoenzymatic coupling is achieved. We found that the interaction of RecQ with DNA results in a drastic acceleration of the rate-limiting ATP cleavage step, which occurs productively due to subsequent rapid phosphate release. ADP release is not rate-limiting and ADP-bound RecQ molecules make up a small fraction during single-stranded DNA translocation. However, the relatively rapid release of the ADP-bound enzyme from DNA causes the majority of translocation run terminations (i.e. detachment from the DNA track). Thus, the DNA interactions of ADP-bound RecQ helicase, probably dependent on DNA structure, will mainly determine translocation processivity and may control the outcome of DNA processing. Comparison with human Bloom''s syndrome (BLM) helicase reveals that similar macroscopic parameters are achieved by markedly different underlying mechanisms of RecQ homologs, suggesting diversity in enzymatic tuning.     

ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase

The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the beta-gamma methylene/amine analogs of ATP, ATPgammaS, or [AlF4]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF3]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3' single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length.     

Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of ∼75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP.     

Bacillus stearothermophilus PcrA monomer is a single-stranded DNA translocase but not a processive helicase in vitro

Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3' to 5' direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.     

DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase.

Based upon the crystal structures of PcrA helicase, we have made and characterised mutations in a number of conserved helicase signature motifs around the ATPase active site. We have also determined structures of complexes of wild-type PcrA with ADPNP and of a mutant PcrA complexed with ADPNP and Mn2+. The kinetic and structural data define roles for a number of different residues in and around the ATP binding site. More importantly, our results also show that there are two functionally distinct conformations of ATP in the active site. In one conformation, ATP is hydrolysed poorly whereas in the other (activated) conformation, ATP is hydrolysed much more rapidly. We propose a mechanism to explain how the stimulation of ATPase activity afforded by binding of single-stranded DNA stabilises the activated conformation favouring Mg2+binding and a consequent repositioning of the gamma-phosphate group which promotes ATP hydrolysis. A part of the associated conformational change in the protein forces the side-chain of K37 to vacate the Mg2+binding site, allowing the cation to bind and interact with ATP.     

Translocation of Escherichia coli recA protein from a single-stranded tail to contiguous duplex DNA

Duplex DNA with a contiguous single-stranded tail was nearly as effective as single-stranded DNA in acting as a cofactor for the ATPase activity of recA protein at neutral pH and concentrations of MgCl2 that support homologous pairing. The ATP hydrolysis reached a steady state rate that was proportional to the length of the duplex DNA attached to a short 5' single-stranded tail after a lag. Separation of the single-stranded tail from most of the duplex portion of the molecule by restriction enzyme cleavage led to a gradual decline in ATP hydrolysis. Measurement of the rate of hydrolysis as a function of DNA concentration for both tailed duplex DNA and single-stranded DNA cofactors indicated that the binding site size of recA protein on a duplex DNA lattice, about 4 base pairs, is similar to that on a single-stranded DNA lattice, about four nucleotides. The length of the lag phase preceding steady state hydrolysis depended on the DNA concentration, length of the duplex region, and the polarity of the single-stranded tail, but was comparatively independent of tail length for tails over 70 nucleotides in length. The lag was 5-10 times longer for 3' than for 5' single-stranded tailed duplex DNA molecules, whereas the steady state rates of hydrolysis were lower. These observations show that, after nucleation of a recA protein complex on the single-stranded tail, the protein samples the entire duplex region via an interaction that is labile and not strongly polarized.     

Mechanism of translocation and kinetics of DNA unwinding by the helicase RecG

RecG is a DNA helicase involved in the repair of damage at a replication fork and catalyzes the reversal of the fork to a point beyond the damage in the template strand. It unwinds duplex DNA in reactions that are coupled to ATP hydrolysis. The kinetic mechanism of duplex DNA unwinding by RecG was analyzed using a quantitative fluorescence assay based on the process of contact quenching between Cy3 and Dabcyl groups attached to synthetic three-way DNA junctions. The data show that the protein moves at a rate of 26 bp s(-1) along the duplex DNA during the unwinding process. RecG ATPase activity during translocation indicates a constant rate of 7.6 s(-1), measured using a fluorescent phosphate sensor, MDCC-PBP. These two rates imply a movement of approximately 3 bp per ATP hydrolyzed. We demonstrate in several trapping experiments that RecG remains attached to DNA after translocation to the end of the arm of the synthetic DNA junction. ATPase activity continues after translocation is complete. Dissociation of RecG from the product DNA occurs only very slowly, suggesting strong interactions between them. The data support the idea that interactions of the duplex template arm with the protein are the major sites of binding and production of translocation.     

RecA protein-promoted cleavage of LexA repressor in the presence of ADP and structural analogues of inorganic phosphate, the fluoride complexes of aluminum and beryllium

Complexes formed from A13+ or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein. In contrast, poly(dT)-RecA-ADP complexes, which are inactive for cleavage of LexA protein, become fully active in the presence of AlF4- or BeF3- ions. These data suggest that fluoride complexes of aluminum and beryllium (called herein X) convert RecA-ADP complexes, which bind weakly to single-stranded DNA, into RecA-ADP-X complexes, which bind tightly to single-stranded DNA, the ADP-X moiety behaving as a nonhydrolyzable analogue of ATP. We propose that AlF4- and BeF3- ions act as analogues of inorganic phosphate by binding to the site of the gamma-phosphate of ATP on RecA-ADP complexes, hence mimicking the single-stranded DNA-RecA-ADP-Pi transition state. We conclude that the elementary reaction that switches RecA protein from a high affinity single-stranded DNA binding state to a low affinity single-stranded DNA binding state is not ATP hydrolysis per se but Pi release.