Kinetic pathway of dTTP hydrolysis by hexameric T7 helicase-primase in the absence of DNA

Bacteriophage T7 gp4A' protein is a hexameric helicase-primase protein that separates the strands of a duplex DNA in a reaction coupled to dTTP hydrolysis. Here we reexamine in more detail the kinetic mechanism of dTTP hydrolysis by a preassembled T7 helicase hexamer in the absence of DNA. Pre-steady state dTTP hydrolysis kinetics showed a distinct burst whose amplitude indicated that a preformed hexamer of T7 helicase hydrolyzes on an average one dTTP per hexamer. The pre-steady state chase-time experiments provided evidence for sequential hydrolysis of two dTTPs. The medium [(18)O]P(i) exchange experiments failed to detect dTTP synthesis, indicating that the less than six-site hydrolysis observed is not due to reversible dTTP hydrolysis on the helicase active site. The P(i)-release rate was measured directly using a stopped-flow fluorescence assay, and it was found that the rate of dTTP hydrolysis on the helicase active site is eight times faster than the P(i)-release rate, which in turn is three times faster than the dTDP release rate. Thus, the rate-limiting step in the pathway of helicase-catalyzed deoxythymidine triphosphatase (dTTPase) reaction is the release of dTDP. Chase-time dTTPase kinetics in the steady state phase provided evidence for two to three slowly hydrolyzing dTTPase sites on the hexamer. The results of this study are therefore consistent with those reported earlier (Hingorani, M. M., Washington, M. T., Moore, K. C., and Patel, S. S. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5012-5017), and they support a model of dTTP hydrolysis by T7 helicase hexamer that is similar to the binding change mechanism of F(1)-ATPase with dTTP hydrolysis occurring sequentially at the catalytic sites.     

On translocation mechanism of ring-shaped helicase along single-stranded DNA

The ring-shaped helicases represent one important group of helicases that can translocate along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA by using the energy derived from NTP binding and hydrolysis. Despite intensive studies, the mechanism by which the ring-shaped helicase translocates along ssDNA and unwinds dsDNA remains undetermined. In order to understand their chemomechanical-coupling mechanism, two models on NTPase activities of the hexamers in the presence of DNA have been studied here. One model is assumed that, of the six nucleotide-binding sites, three are noncatalytic and three are catalytic. The other model is assumed that all the six nucleotide-binding sites are catalytic. In terms of the sequential NTPase activity around the ring and the previous determined crystal structure of bacteriophage T7 helicase it is shown that the obtained mechanical behaviors such as the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle using the former model are in good quantitative agreement with the previous experimental results for T7 helicase. Moreover, the acceleration of DNA unwinding rate with the stimulation of DNA synthesis by DNA polymerase can also be well explained by using the former model. In contrast, the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle obtained by using the latter model are not consistent with the experimental results for T7 helicase. Thus it is preferred that the former model is the appropriate one for the T7 helicase. Furthermore, using the former model some dynamic behaviors such as the rotational speeds of DNA relative to the T7 helicase when translocation along ssDNA and when unwinding dsDNA have been predicted, which are expected to test in order to further verify the model.     

Mechanisms of a ring shaped helicase

Bacteriophage T7 helicase (T7 gene 4 helicase-primase) is a prototypical member of the ring-shaped family of helicases, whose structure and biochemical mechanisms have been studied in detail. T7 helicase assembles into a homohexameric ring that binds single-stranded DNA in its central channel. Using RecA-type nucleotide binding and sensing motifs, T7 helicase binds and hydrolyzes several NTPs, among which dTTP supports optimal protein assembly, DNA binding and unwinding activities. During translocation along single stranded DNA, the subunits of the ring go through dTTP hydrolysis cycles one at a time, and this probably occurs also during DNA unwinding. Interestingly, the unwinding speed of T7 helicase is an order of magnitude slower than its translocation rate along single stranded DNA. The slow unwinding rate is greatly stimulated when DNA synthesis by T7 DNA polymerase is coupled to DNA unwinding. Using the T7 helicase as an example, we highlight critical findings and discuss possible mechanisms of helicase action.     

Coupling of DNA unwinding to nucleotide hydrolysis in a ring-shaped helicase

The ring-shaped T7 helicase uses the energy of dTTP hydrolysis to perform the mechanical work of translocation and base pair (bp) separation. We have shown that the unwinding rate of T7 helicase decreases with increasing DNA stability. Here, we show that the dTTPase rate also decreases with increasing DNA stability, which indicates close linkage between chemical transition steps and translocation steps of unwinding. We find that the force-producing step during unwinding is not associated with dTTP binding, but dTTP hydrolysis or P(i) release. We determine that T7 helicase extracts approximately 3.7 kcal/mol energy from dTTPase to carry out the work of strand separation. Using this energy, T7 helicase unwinds approximately 4 bp of AT-rich DNA or 1-2 bp of GC-rich DNA. T7 helicase therefore adjusts both its speed and coupling ratio (bp/dTTP) to match the work of DNA unwinding. We discuss the mechanistic implications of the variable bp/dTTP that indicates T7 helicase either undergoes backward movements/futile hydrolysis or unwinds DNA with a variable bp-step size; 'long and fast' steps on AT-rich and 'short and slow' steps on GC-rich 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.     

Unraveling DNA helicases. Motif, structure, mechanism and function.

DNA helicases are molecular 'motor' enzymes that use the energy of NTP hydrolysis to separate transiently energetically stable duplex DNA into single strands. They are therefore essential in nearly all DNA metabolic transactions. They act as essential molecular tools for the cellular machinery. Since the discovery of the first DNA helicase in Escherichia coli in 1976, several have been isolated from both prokaryotic and eukaryotic systems. DNA helicases generally bind to ssDNA or ssDNA/dsDNA junctions and translocate mainly unidirectionally along the bound strand and disrupt the hydrogen bonds between the duplexes. Most helicases contain conserved motifs which act as an engine to drive DNA unwinding. Crystal structures have revealed an underlying common structural fold for their function. These structures suggest the role of the helicase motifs in catalytic function and offer clues as to how these proteins can translocate and unwind DNA. The genes containing helicase motifs may have evolved from a common ancestor. In this review we cover the conserved motifs, structural information, mechanism of DNA unwinding and translocation, and functional aspects of DNA helicases.     

DNA helicases displace streptavidin from biotin-labeled oligonucleotides

Helicases are enzymes that use energy derived from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. The helicases used in this study, gp41 and Dda, are from the bacteriophage T4, an excellent system for studying enzymes that process DNA. gp41 is the replicative helicase and has been shown to form a hexamer in the presence of ATP. In this study, protein cross-linking was performed in the presence of either linear or circular single-stranded (ss) DNA substrates to determine the topology of gp41 binding to ssDNA. Results indicate that the hexamer binds ssDNA by encircling it, in a manner similar to that of other hexameric helicases. A new assay was developed for studying enzymatic activity of gp41 and Dda on single-stranded DNA. The rate of dissociation of streptavidin from various biotinylated oligonucleotides was determined in the presence of helicase by an electrophoretic mobility shift assay. gp41 and Dda were found to significantly enhance the dissociation rate of streptavidin from biotin-labeled oligonucleotides in an ATP-dependent reaction. Helicase-catalyzed dissociation of streptavidin from the 3'-end of a biotin-labeled 62-mer oligonucleotide occurred with a first-order rate of 0.17 min-1, which is over 500-fold faster than the spontaneous dissociation rate of biotin from streptavidin. Dda activity leads to even faster displacement of streptavidin from the 3' end of the 62-mer, with a first-order rate of 7.9 s-1. This is more than a million-fold greater than the spontaneous dissociation rate. There was no enhancement of streptavidin dissociation from the 5'-biotin-labeled oligonucleotide by either helicase. The fact that each helicase was capable of dislodging streptavidin from the 3'-biotin label suggests that these enzymes are capable of imparting a force on a molecule blocking their path. The difference in displacement between the 5' and 3' ends of the oligonucleotide is also consistent with the possibility of a 5'-to-3' directional bias in translocation on ssDNA for each helicase.     

Translocation of E. coli RecQ helicase on single-stranded DNA

A member of the SF2 family of helicases, Escherichia coli RecQ, is involved in the recombination and repair of double-stranded DNA breaks and single-stranded DNA (ssDNA) gaps. Although the unwinding activity of this helicase has been studied biochemically, the mechanism of translocation remains unclear. To this end, using ssDNA of varying lengths, the steady-state ATP hydrolysis activity of RecQ was analyzed. We find that the rate of ATP hydrolysis increases with DNA length, reaching a maximum specific activity of 38 ± 2 ATP/RecQ/s. Analysis of the rate of ATP hydrolysis as a function of DNA length implies that the helicase has a processivity of 19 ± 6 nucleotides on ssDNA and that RecQ requires a minimal translocation site size of 10 ± 1 nucleotides. Using the T4 phage encoded gene 32 protein (G32P), which binds ssDNA cooperatively, to decrease the lengths of ssDNA gaps available for translocation, we observe a decrease in the rate of ATP hydrolysis activity that is related to lattice occupancy. Analysis of the activity in terms of the average gap sizes available to RecQ on the ssDNA coated with G32P indicates that RecQ translocates on ssDNA on average 46 ± 11 nucleotides before dissociating. Moreover, when bound to ssDNA, RecQ hydrolyzes ATP in a cooperative fashion, with a Hill coefficient of 2.1 ± 0.6, suggesting that at least a dimer is required for translocation on ssDNA. We present a kinetic model for translocation by RecQ on ssDNA based on this characterization.     

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.     

UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke

Helicases use the energy derived from nucleoside triphosphate hydrolysis to unwind double helices in essentially every metabolic pathway involving nucleic acids. Earlier crystal structures have suggested that DNA helicases translocate along a single-stranded DNA in an inchworm fashion. We report here a series of crystal structures of the UvrD helicase complexed with DNA and ATP hydrolysis intermediates. These structures reveal that ATP binding alone leads to unwinding of 1 base pair by directional rotation and translation of the DNA duplex, and ADP and Pi release leads to translocation of the developing single strand. Thus DNA unwinding is achieved by a two-part power stroke in a combined wrench-and-inchworm mechanism. The rotational angle and translational distance of DNA define the unwinding step to be 1 base pair per ATP hydrolyzed. Finally, a gateway for ssDNA translocation and an alternative strand-displacement mode may explain the varying step sizes reported previously.     

Model for helicase translocating along single-stranded DNA and unwinding double-stranded DNA

A model is proposed for non-hexameric helicases translocating along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA. The translocation of a monomeric helicase along ssDNA in weakly-ssDNA-bound state is driven by the Stokes force that is resulted from the conformational change following the transition of the nucleotide state. The unwinding of dsDNA is resulted mainly from the bending of ssDNA induced by the strong binding force of helicase with dsDNA. The interaction force between ssDNA and helicases in weakly-ssDNA-bound state determines whether monomeric helicases such as PcrA can unwind dsDNA or dimeric helicases such as Rep are required to unwind dsDNA.     

Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2-4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.     

Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase

Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V_un) by the velocity of translocation on ssDNA in nucleotides per second (V_trans). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the “active” range. In the case of Dda, the average DNA unwinding velocity was 257 ± 42 bp/s, and the average translocation velocity was 267 ± 15 nt/s. The V_un/V_trans value of 0.96 places Dda in a unique category of being an essentially “perfectly” active helicase.     

The linker region between the helicase and primase domains of the gene 4 protein of bacteriophage T7. Role in helicase conformation and activity

The gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for template-directed oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246-271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala257, Pro259, and Asp263) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4. The in vivo screening revealed Ala257 and Asp263 to be essential whereas Pro259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala257 or Asp263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg2+, all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer.     

The T4 phage SF1B helicase Dda is structurally optimized to perform DNA strand separation

Helicases move on DNA via an ATP binding and hydrolysis mechanism coordinated by well-characterized helicase motifs. However, the translocation along single-stranded DNA (ssDNA) and the strand separation of double-stranded (dsDNA) may be loosely or tightly coupled. Dda is a phage T4 SF1B helicase with sequence homology to the Pif1 family of helicases that tightly couples translocation to strand separation. The crystal structure of the Dda-ssDNA binary complex reveals a domain referred to as the "pin" that was previously thought to remain static during strand separation. The pin contains a conserved phenylalanine that mediates a transient base-stacking interaction that is absolutely required for separation of dsDNA. The pin is secured at its tip by protein-protein interactions through an extended SH3 domain thereby creating a rigid strut. The conserved interface between the pin and the SH3 domain provides the mechanism for tight coupling of translocation to strand separation.     

Benzo[a]pyrene-DNA adducts inhibit translocation by the gene 4 protein of bacteriophage T7

Bacteriophage T7 gene 4 protein is an essential component of the T7 DNA replication system, acting as both a primase and a helicase. The gene 4 protein has been shown to translocate along single-stranded DNA in the 5'----3' direction, using an energy source for this movement the hydrolysis of nucleoside 5'-triphosphates, preferably dTTP. Thus, measuring the rate and extent of dTTP hydrolysis provides a means to directly measure translocation. We have determined that the hydrolysis of dTTP by the gene 4 protein is strongly inhibited by the presence of benzo[a]pyrene (B[a]P) adducts on the DNA. Time course experiments on adduct-containing DNA show that after an initial burst of hydrolysis, which parallels what is observed on unmodified DNA, further hydrolysis abruptly ceases. Addition of excess unmodified DNA does not restore the hydrolysis activity. These data suggest that the gene 4 protein is blocked and sequestered on the DNA at the site of the adduct. This was confirmed by experiments in which gene 4 protein preferentially protected the radiolabeled adduct-containing DNA but not randomly labeled M13 DNA. The gene 4 protein bound to the B[a]P-modified DNA was isolated, and the complex was found only to contain dTTP. These results have been used to formulate a model for gene 4 protein translocation in which we speculate that the power stroke for unidirectional movement along the single-stranded DNA is the displacement of dTDP by dTTP. Finally, we observe a constant ratio of DNA synthesis to dTTP hydrolysis regardless of the number of B[a]P adducts in the template suggesting that a significant portion of the inhibition of DNA synthesis is a direct consequence of the inhibition of gene 4 translocation.     

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.     

Modular architecture of the hexameric human mitochondrial DNA helicase

We have probed the structure of the human mitochondrial DNA helicase, an enzyme that uses the energy of nucleotide hydrolysis to unwind duplex DNA during mitochondrial DNA replication. This novel helicase shares substantial amino acid sequence and functional similarities with the bacteriophage T7 primase-helicase. We show in velocity sedimentation and gel filtration analyses that the mitochondrial DNA helicase exists as a hexamer. Limited proteolysis by trypsin results in the production of several stable fragments, and N-terminal sequencing reveals distinct N and C-terminal polypeptides that represent minimal structural domains. Physical analysis of the proteolytic products defines the region required to maintain oligomeric structure to reside within amino acid residues approximately 405-590. Truncations of the N and C termini affect differentially DNA-dependent ATPase activity, and whereas a C-terminal domain polypeptide is functional, an N-terminal domain polypeptide lacks ATPase activity. Sequence similarity and secondary structural alignments combined with biochemical data suggest that amino acid residue R609 serves as the putative arginine finger that is essential for ATPase activity in ring helicases. The hexameric conformation and modular architecture revealed in our study document that the mitochondrial DNA helicase and bacteriophage T7 primase-helicase share physical features. Our findings place the mitochondrial DNA helicase firmly in the DnaB-like family of replicative DNA helicases.     

Promiscuous Usage of Nucleotides by the DNA Helicase of Bacteriophage T7: DETERMINANTS OF NUCLEOTIDE SPECIFICITY*S?

The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.Helicases are molecular machines that translocate unidirectionally along single-stranded nucleic acids using the energy derived from nucleotide hydrolysis (1–3). The gene 4 protein encoded by bacteriophage T7 consists of a helicase domain and a primase domain, located in the C-terminal and N-terminal halves of the protein, respectively (4). The T7 helicase functions as a hexamer and has been used as a model to study ring-shaped replicative helicases. In the presence of dTTP, T7 helicase binds to single-stranded DNA (ssDNA)³ as a hexamer and translocates 5′ to 3′ along the DNA strand using the energy of hydrolysis of dTTP (5–7). T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; however, dTTP hydrolysis is optimally coupled to DNA unwinding (5).Most hexameric helicases use rATP to fuel translocation and unwind DNA (3). T7 helicase does hydrolyze rATP but with a 20-fold higher K_m as compared with dTTP (5, 8). It has been suggested that T7 helicase actually uses rATP in vivo where the concentration of rATP is 20-fold that of dTTP in the Escherichia coli cell (8). However, hydrolysis of rATP, even at optimal concentrations, is poorly coupled to translocation and unwinding of DNA (9). Other ribonucleotides (rCTP, rGTP, and rUTP) are either not hydrolyzed or the poor hydrolysis observed is not coupled to DNA unwinding (8). Furthermore, Patel et al. (10) found that the form of T7 helicase found in vivo, an equimolar mixture of the full-length gp4 and a truncated form lacking the zinc binding domain of the primase, prefers dTTP and dATP. Therefore, in the present study we have restricted our examination of nucleotides to the deoxyribonucleotides.The nucleotide binding site of the replicative DNA helicases, such as T7 gene 4 protein, bind nucleotides at the subunit interface (Fig. 1) located between two RecA-like subdomains that bind ATP (11, 12). The location of the nucleotide binding site at the subunit interface provides multiple interactions of residues with the bound NTP. A number of cis- and trans-acting amino acids stabilize the bound nucleotide in the nucleotide binding site and also provide for communication between subunits (13–15). Earlier reports revealed that the arginine finger (Arg-522) in T7 helicase is positioned to interact with the γ-phosphate of the bound nucleotide in the adjacent subunit (12, 16). However, His-465 (phosphate sensor), Glu-343 (catalytic base), and Asp-424 (Walker motif B) interacts with the γ-phosphate of the bound nucleotide in the same subunit (12, 17, 18). The arginine finger and the phosphate sensor have been proposed to couple NTP hydrolysis to DNA unwinding. Substitution of Glu-343, the catalytic base, eliminates dTTP hydrolysis (19), and substitution of Asp-424 with Asn leads to a severe reduction in dTTP hydrolysis (20). The conserved Lys-318 in Walker motif A interacts with the β-phosphate of the bound nucleotide and plays an important role in dTTP hydrolysis (21).Open in a separate windowFIGURE 1.Crystal structure of T7 helicase. A, crystal structure of the hexameric helicase C-terminal domain of gp4 (17). The structure reveals a ring-shaped molecule with a central core through which ssDNA passes. The inset shows the interface between two subunits of the helicase with adenosine 5′-{β,γ-imidol}-triphosphate in the nucleotide binding site. B, the nucleotide binding site of a monomer of the gp4 with the crucial amino acid residues reported earlier and in the present study is shown in sticks. The crystal structures of the T7 gene 4 helicase domain (12) with bound dTTP (C) and dATP (D). The structures shown are the nucleotide binding site of T7 helicase as viewed in Pymol by analyzing the PDB files 1cr1 and 1cr2 (12). Arg-504 and Tyr-535 sandwiches the base of the bound dNTP. Additionally, Arg-504 forms a hydrogen bridge with dTTP. Arg-363 interacts specifically with the 3-OH group of bound dATP. AMPPNP, adenosine 5′-(β,γ-imino)triphosphate.Considering the wealth of information on the above residues that are involved in the hydrolysis of dTTP and the coupling of hydrolysis to unwinding, it is intriguing that little information is available on nucleotide specificity. Several crystal structures of T7 helicase in complex with a nucleotide triphosphate are available. However, most of structures were crystallized with a non-hydrolyzable analogue of dTTP or the nucleotide was diffused into the crystal. The crystal structure of the T7 helicase domain bound with dTTP or dATP was reported by Sawaya et al. (12). These structures assisted us in identifying two basic residues (Arg-363 and Arg-504) in close proximity to the sugar and base of the bound nucleotide whose orientation suggested that these residues could be involved in nucleotide selection. Arg-504 together with Tyr-535 sandwich the base of the bound nucleotide at the subunit interface of the hexameric helicase (Fig. 1). Arg-504 and Tyr-535 are structurally well conserved in various helicases (12). However, Arg-504 could make a hydrogen bridge with the OH group of thymidine, thus suggesting a role in dTTP specificity. On the other hand, Arg-363 is in close proximity (∼3.4 Å) to the sugar 3′-OH of bound dATP, whereas in the dTTP-bound structure this residue is displaced by 7.12 Å (Fig. 1) from the equivalent position. Consequently Arg-363 could play a role in dATP binding. The crystal structures do not provide any information on different interaction of residues with the phosphates of dATP and dTTP. However, alignment of the residues in the P-loops of different hexameric helicases reveals that the serine adjacent to the invariant lysine at position 319 (Ser-319) is conserved in bacteriophages, whereas bacterial helicases have a conserved threonine in the equivalent position (supplemental Fig. 1). Bacterial helicases use rATP in the DNA unwinding reactions. whereas T7 helicase preferentially uses dTTP, and bacteriophage T4 gene 41 uses rGTP or rATP (22).Although considerable information is available on the role of residues in nucleotide binding and dTTP hydrolysis, very little is known on the determinants of nucleotide specificity. In the present study we made an attempt to address the role of a few selected residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity, especially dTTP and dATP, both of which are hydrolyzed and mediate DNA unwinding. We show that under physiological conditions T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.