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.     

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.     

Production of two variant laminin forms by endothelial cells and shift of their relative levels by angiostatic steroids.

Organization of endothelium as the lining of the cardiovascular system is supported by basement membrane. The important role of laminin and other basement membrane proteins is assumed in the angiogenesis. We show here that cultured endothelial cells produce two forms of laminin, and their relative levels are changed by antiangiogenic steroids. The synthesis of laminin subunits by endothelial cells isolated from bovine aorta and from bovine pulmonary artery was studied by metabolic labeling with [35S]methionine. Both endothelial cells produced a novel laminin-related polypeptide (A' subunit) in addition to the A, B1, and B2 subunits. Two-dimensional sodium dodecyl sulfate gel electrophoretic analysis showed that the B1B2 complex was first formed and the A subunit joined it to form the AB1B2 complex or the A' subunit joined it to form A'B1B2 complex. This mechanism implied that replacement of subunits in the complex by a corresponding variant produces variety in the structure and function of laminin. The A'B1B2 complex was the major product in endothelial cells under normal culture conditions. An angiostatic steroid, medroxyprogesterone, suppressed the A' synthesis and stimulated the A synthesis. Consequently, the major product of bovine aorta endothelial cells was converted to AB1B2. Two types of intracellular precursors were identified for each laminin-related polypeptide. Since the precursors in a given complex were synchronized with regard to maturation, the assembly of AB1B2 and A'B1B2 complexes was suggested to occur at an early step of intracellular processing.     

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.     

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.     

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.     

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.     

Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase.

T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon. These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells. They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein. Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth. Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity. The mutation at amino acid 64 of 4A' appears to have little effect on 4A function. The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth.     

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.