Nucleotide-dependent binding of the gene 4 protein of bacteriophage T7 to single-stranded DNA

The gene 4 protein of bacteriophage T7 is a multifunctional enzyme that catalyzes (i) the hydrolysis of nucleoside 5'-triphosphates, (ii) the synthesis of tetraribonucleotide primers at specific recognition sequences on a DNA template, and (iii) the unwinding of duplex DNA. All three activities depend on binding of gene 4 protein to single-stranded DNA followed by unidirectional 5' to 3' translocation of the protein (Tabor, S., and Richardson, C. C. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 205-209). Binding of gene 4 protein to single-stranded DNA, assayed by retention of DNA-protein complexes on nitrocellulose filters, is random with regard to DNA sequence. Although gene 4 protein does not bind to duplex DNAs, the presence of a 240-nucleotide-long single-stranded tail on a 7200-base pair duplex DNA molecule is sufficient for gene 4 protein to cause retention of the DNA on a filter. The binding reaction requires, in addition to MgCl2, the presence of a nucleoside 5'-triphosphate, but binding is not dependent on hydrolysis; nucleoside 5'-diphosphate will substitute for nucleoside 5'-triphosphate. Of the eight common nucleoside triphosphates, dTTP promotes optimal binding. The half-life of the gene 4 protein-DNA complex depends on both the secondary structure of the DNA and on whether or not the nucleoside 5'-triphosphate cofactor can be hydrolyzed. Using the nonhydrolyzable nucleoside 5'-triphosphate analog, beta,gamma-methylene dTTP, the half-life of the gene 4 protein-DNA complex is greater than 80 min. In the presence of the hydrolyzable nucleoside 5'-triphosphate, dTTP, the half-life of the gene 4 protein-DNA complex using circular M13 DNA is at least 4 times longer than that observed using linear M13 DNA.     

Purification of the 56-kDa component of the bacteriophage T7 primase/helicase and characterization of its nucleoside 5'-triphosphatase activity

Bacteriophage T7 gene 4 protein, purified from phage-infected cells, consists of a mixture of a 56- and a 63-kDa species that provides primase and helicase activities for T7 DNA replication. The 56-kDa species has been purified 1800-fold from Escherichia coli cells containing a plasmid that encodes this gene 4 protein. The purified 56-kDa protein is homogeneous, as determined by denaturing gel electrophoresis, and is monomeric in its native form, as indicated by gel filtration. The binding of the 56-kDa protein to single-stranded DNA is stimulated by nucleoside 5'-triphosphates, as is the case for a mixture of the two molecular weight species. In the presence of DNA, the 56-kDa protein preferentially hydrolyzes dTTP (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 396-400). Since nucleoside 5'-triphosphatase activity is necessary for both helicase activity and for translocation of gene 4 protein to primase recognition sites, we have characterized this activity using the 56-kDa protein alone. In the DNA-dependent hydrolysis reaction, the enzyme displays a Km of 10 mM for dTTP, and a Vmax of 2.9 x 10(-5) M/min/mg of protein (at 2.5 micrograms/ml). There is little cooperativity with respect to dTTP binding (Hill coefficient = 1.1) except in the presence of ribonucleoside 5'-triphosphate, an inhibitor of dTTP hydrolysis (Hill coefficient greater than 1.5). The apparent KD for single-stranded circular DNA is 0.2 microM. The active species in dTTP hydrolysis is an oligomer of at least two subunits, as indicated by the effect of enzyme concentration upon the rate of DNA-dependent hydrolysis. The 56-kDa protein also catalyzes DNA-independent hydrolysis of dTTP with a Km of 0.11 mM and a Vmax of 1.3 x 10(-7) M/min/mg of protein (at 8 micrograms/ml). The active species in DNA-independent dTTP hydrolysis is also an oligomer.     

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.     

The C-terminal residues of bacteriophage T7 gene 4 helicase-primase coordinate helicase and DNA polymerase activities

The gene 4 protein of bacteriophage T7 plays a central role in DNA replication by providing both helicase and primase activities. The C-terminal helicase domain is not only responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding, but it also interacts with T7 DNA polymerase to coordinate helicase and polymerase activities. The C-terminal 17 residues of gene 4 protein are critical for its interaction with the T7 DNA polymerase/thioredoxin complex. This C terminus is highly acidic; replacement of these residues with uncharged residues leads to a loss of interaction with T7 DNA polymerase/thioredoxin and an increase in oligomerization of the gene 4 protein. Such an alteration on the C terminus results in a reduced efficiency in strand displacement DNA synthesis catalyzed by gene 4 protein and T7 DNA polymerase/thioredoxin. Replacement of the C-terminal amino acid, phenylalanine, with non-aromatic residues also leads to a loss of interaction of gene 4 protein with T7 DNA polymerase/thioredoxin. However, neither of these modifications of the C terminus affects helicase and primase activities. A chimeric gene 4 protein containing the acidic C terminus of the T7 gene 2.5 single-stranded DNA-binding protein is more active in strand displacement synthesis. Gene 4 hexamers containing even one subunit of a defective C terminus are defective in their interaction with T7 DNA polymerase.     

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.     

Characterization of the helicase and primase activities of the 63-kDa component of the bacteriophage T7 gene 4 protein

Leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7 DNA requires the helicase and primase activities of the gene 4 protein. Gene 4 protein consists of two colinear polypeptides of 56- and 63-kDa molecular mass. We have demonstrated previously that the 56-kDa protein possesses helicase but lacks primase activity (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 396-400). The 63-kDa gene 4 protein has now been purified from extracts of T7-infected cells. The preparation contains 5-10% contaminating 56-kDa protein, as shown by Western analysis using polyclonal antibodies to the purified 56-kDa protein. The 63-kDa protein catalyzes DNA-dependent dTTP hydrolysis and has helicase activity; both specific activities are similar to those determined for the 56-kDa protein. The 63-kDa protein efficiently synthesizes sequence-specific di-, tri-, and tetraribonucleotides and stimulates the elongation of tetraribonucleotides by T7 DNA polymerase. Although the 56-kDa protein alone lacks primase activity, it enhances the primase activity of the 63-kDa protein 4-fold. This stimulation can be accounted for by a similar increase in the amount of primers synthesized by the 63-kDa protein in the presence of the 56-kDa protein.     

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site.

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.     

Effect of single-stranded DNA-binding proteins on the helicase and primase activities of the bacteriophage T7 gene 4 protein

Gene 4 protein (gp4) of bacteriophage T7 provides two essential functions at the T7 replication fork, primase and helicase activities. Previous studies have shown that the single-stranded DNA-binding protein of T7, encoded by gene 2.5, interacts with gp4 and modulates its multiple functions. To further characterize the interactions between gp4 and gene 2.5 protein (gp2.5), we have examined the effect of wild-type and altered gene 2.5 proteins as well as Escherichia coli single-stranded DNA-binding (SSB) protein on the ability of gp4 to synthesize primers, hydrolyze dTTP, and unwind duplex DNA. Wild-type gp2.5 and E. coli SSB protein stimulate primer synthesis and DNA-unwinding activities of gp4 at low concentrations but do not significantly affect single-stranded DNA-dependent hydrolysis of dTTP. Neither protein inhibits the binding of gp4 to single-stranded DNA. The variant gene 2.5 proteins, gp2.5-F232L and gp2.5-Delta26C, inhibit primase, dTTPase, and helicase activities proportional to their increased affinities for DNA. Interestingly, wild-type gp2.5 stimulates the unwinding activity of gp4 except at very high concentrations, whereas E. coli SSB protein is highly inhibitory at relative low concentrations.     

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.     

Oligomeric states of bacteriophage T7 gene 4 primase/helicase

Electron microscopic and crystallographic data have shown that the gene 4 primase/helicase encoded by bacteriophage T7 can form both hexamers and heptamers. After cross-linking with glutaraldehyde to stabilize the oligomeric protein, hexamers and heptamers can be distinguished either by negative stain electron microscopy or electrophoretic analysis using polyacrylamide gels. We find that hexamers predominate in the presence of either dTTP or beta,gamma-methylene dTTP whereas the ratio between hexamers and heptamers is nearly the converse in the presence of dTDP. When formed, heptamers are unable to efficiently bind either single-stranded DNA or double-stranded DNA. We postulate that a switch between heptamer to hexamer may provide a ring-opening mechanism for the single-stranded DNA binding pathway. Accordingly, we observe that in the presence of both nucleoside di- and triphosphates the gene 4 protein exists as a hexamer when bound to single-stranded DNA and as a mixture of heptamer and hexamer when not bound to single-stranded DNA. Furthermore, altering regions of the gene 4 protein postulated to be conformational switches for dTTP-dependent helicase activity leads to modulation of the heptamer to hexamer ratio.     

DNA-induced switch from independent to sequential dTTP hydrolysis in the bacteriophage T7 DNA helicase

We show that the mechanisms of DNA-dependent and -independent dTTP hydrolysis by the gene 4 protein of bacteriophage T7 differ in the pathways by which these reactions are catalyzed. In the presence of dTTP, gene 4 protein monomers assemble as a ring that binds single-stranded DNA and couples the hydrolysis of dTTP to unidirectional translocation and the unwinding of duplex DNA. When mixing wild-type monomers with monomers lacking the catalytic base for the dTTPase reaction, we observe that each wild-type subunit hydrolyzes dTTP independently in the absence of single-stranded DNA. Conversely, when either these catalytically inactive monomers or altered monomers incapable of binding single-stranded DNA are mixed with wild-type monomers, a small fraction of altered to wild-type monomers causes a sharp decline in DNA-dependent dTTP hydrolysis. We propose that sequential hydrolysis of dTTP is coupled to the transfer of single-stranded DNA from subunit to adjacent subunit.     

Escherichia coli helicase II (urvD gene product) translocates unidirectionally in a 3' to 5' direction

Escherichia coli helicase II, product of the uvrD gene, is a single-stranded DNA-dependent nucleoside 5'-triphosphatase with helicase activity. As a DNA-dependent ATPase, helicase II translocates processively along single-stranded DNA (S. W. Matson, unpublished results). The direction of translocation has been determined using a helicase assay that directly measures the ability of helicase II to catalyze the displacement of a labeled DNA fragment from one end of a single-stranded linear DNA molecule. The translocation of helicase II along single-stranded DNA is unidirectional and in the 3' to 5' direction with respect to the DNA strand on which the enzyme is bound. A kinetic analysis of the displacement of a labeled DNA fragment annealed to a linear single-stranded DNA molecule is also consistent with unidirectional translocation in the 3' to 5' direction. These results are contrary to results previously obtained using an indirect helicase assay (Kuhn, B., Abdel-Monem, M., Krell, H., and Hoffmann-Berling, H. (1979) J. Biol. Chem. 254, 11343-11350).     

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.     

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.     

Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 protein.

Gene 4 of bacteriophage T7 encodes two proteins, a 63-kDa protein and a colinear 56-kDa protein, that are essential for synthesis of leading and lagging strands during DNA replication. The gene 4 proteins together catalyze the synthesis of oligoribonucleotides, pppACC(C/A) or pppACAC, at the single-stranded DNA sequences 3'-CTGG(G/T)-5' or 3'-CTGTG-5', respectively. Purified 56-kDa protein has helicase activity, but no primase activity. In order to study 63-kDa gene 4 protein free of 56-kDa gene 4 protein, mutations were introduced into the internal ribosome-binding site responsible for the translation of the 56-kDa protein. The 63-kDa gene 4 protein was purified 16,000-fold from Escherichia coli cells harboring an expression vector containing the mutated gene 4. Purified 63-kDa gene 4 protein has primase, helicase, and single-stranded DNA-dependent dTTPase activities. The constraints of primase recognition sequences, nucleotide substrate requirements, and the effects of additional proteins on oligoribonucleotide synthesis by the 63-kDa gene 4 protein have been examined using templates of defined sequence. A three-base sequence, 3'-CTG-5', is necessary and sufficient to support the synthesis of pppAC dimers. dTTP hydrolysis is essential for oligoribonucleotide synthesis. Addition of a 7-fold molar excess of 56-kDa gene 4 protein to 63-kDa protein increases the number of oligoribonucleotides synthesized by 63-kDa protein 100-fold. The increase in oligonucleotides results predominantly from an increase in the synthesis of tetramers, with relatively little change in the synthesis of dimers and trimers. The presence of 56-kDa protein also causes 63-kDa protein to synthesize "pseudo-templated" pppACCCC pentamers at the recognition sequence 3'-CTGGG-5'. T7 gene 2.5 protein, a single-stranded DNA binding protein, increases the total number of oligoribonucleotides synthesized by 63-kDa gene 4 protein on single-stranded M13 DNA, but has no effect on the ratio of dimers to trimers and tetramers.     

Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation.

A rapid purification procedure produces milligram amounts of the T7 gene 4A' primase/helicase, 4B helicase, and the wild-type 4AB proteins expressed from the clones described in the accompanying paper (Rosenberg, A. H., Patel, S. S., Johnson, K. A., and Studier, F. W. (1992) J. Biol. Chem. 267, 15005-15012). Purified 4A' protein (in which the wild-type methionine at amino acid 64 has been replaced by leucine to eliminate the 4B initiation codon) appears to be equivalent to the wild-type 4A protein in primase, helicase, and NTPase activities. Gel filtration chromatography and polyacrylamide gel electrophoresis of native proteins indicate that the 4A' and 4B proteins form homodimers and heterodimers in solution. Heterodimer formation presumably accounts for an observed 3-fold increase in the primase activity of 4A' upon addition of 4B that lacks primase activity of its own. Steady-state k(cat) and Km values for hydrolysis of the nucleoside triphosphates ATP, dATP, dTTP, and dGTP were measured for 4A', 4B, 4A'B (1:1), and wild-type 4AB (1:2) proteins. The dependence of the dNTPase activities on the concentration was hyperbolic, suggesting single or noncooperative binding sites, whereas ATPase activity was sigmoidal, suggesting more than one ATP binding site. The k(cat)/Km ratios for hydrolysis of the dNTPs by the four protein preparations were within a factor of 6 of each other. The 1:1 mixture of 4A'B had the highest k(cat)/Km ratios, with a preference for dATP and dTTP.     

The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation.

The gene 4 protein of bacteriophage T7, a functional hexamer, comprises DNA helicase and primase activities. Both activities depend on the unidirectional movement of the protein along single-stranded DNA in a reaction coupled to the hydrolysis of dTTP. We have characterized dTTPase activity and hexamer formation for the full-length gene 4 protein (gp4) as well as for three carboxyl-terminal fragments starting at residues 219 (gp4-C219), 241 (gp4-C241), and 272 (gp4-C272). The region between residues 242 and 271, residing between the primase and helicase domains, is critical for oligomerization of the gene 4 protein. A functional TPase active site is dependent on oligomerization. During native gel electrophoresis, gp4, gp4-C219, and gp4-C241 migrate as oligomers, whereas gp4-C272 is monomeric. The steady-state k(cat) for dTTPase activity of gp4-C272 increases sharply with protein concentration, indicating that it forms oligomers only at high concentrations. gp4-C219 and gp4-C241 both form a stable complex with gp4, whereas gp4-C272 interacts only weakly with gp4. Measurements of surface plasmon resonance indicate that a monomer of T7 DNA polymerase binds to a dimer of gp4, gp4-C219, or gp4-C241 but to a monomer of gp4-C272. Like the homologous RecA and F(1)-ATPase proteins, the oligomerization domain of the gene 4 protein is adjacent to the amino terminus of the NTP-binding domain.     

Phage P4 alpha protein is multifunctional with origin recognition, helicase and primase activities.

alpha Protein of satellite phage P4 of Escherichia coli is multifunctional in P4 replication with three activities. First, the protein (subunit M(r) = 84,900) complexes specifically the P4 origin and the cis replication region required for replication. alpha Protein interacts with all six type I repeats (TGTTCACC) present in the origin. Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate. All common NTPs except UTP and dTTP can serve as cofactors. Strand separation of partial duplexes containing tailed ends that resemble a replication fork is preferred, although a preformed fork is not absolutely required for the enzyme to invade and unwind duplex DNA. alpha Protein catalyzes unwinding in the 3'-5' direction with respect to the strand it has bound. Finally, the primase activity already demonstrated for alpha protein is due to synthesis of RNA primers. In vitro, alpha protein generates di- to pentaribonucleotides on single-stranded phage fd DNA. The predominant product is the dimer pppApG, on which most of the longer oligoribonucleotides are based. Using DNA oligonucleotides of defined sequence as templates, synthesis of pppApG was also detectable. To date, among prokaryotic and eukaryotic replication systems, gp alpha is the only protein known that combines three activities on one single polypeptide chain.     

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.     

The primase active site is on the outside of the hexameric bacteriophage T7 gene 4 helicase-primase ring

Gene 4 of bacteriophage T7 encodes a protein (gp4) that can translocate along single-stranded DNA, couple the unwinding of duplex DNA with the hydrolysis of dTTP, and catalyze the synthesis of short RNA oligoribonucleotides for use as primers by T7 DNA polymerase. Electron microscopic studies have shown that gp4 forms hexameric rings, and X-ray crystal structures of the gp4 helicase domain and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined. Earlier biochemical studies have shown that when single-stranded DNA is bound to the hexameric ring, the primase domain remains accessible to free DNA. Given these results, a model was suggested in which the primase active site in the gp4 hexamer is located on the outside of the hexameric ring. We have used electron microscopy and single-particle image analysis to examine T7 gp4, and have determined that the primase active site is located on the outside of the hexameric ring, and therefore provide direct structural support for this model.