The Replication System of Bacteriophage T7.

The replication system of bacteriophage T7 is remarkable in that the 40,000 nucleotide genome is replicated over 100-fold in a matter of minutes. In order to accomplish this feat T7 has evolved an efficient and economical process for the replication of its DNA. The T7 replisome provides a model system to study DNA replication. Four proteins are sufficient for reconstitution of the functional replication complex, yet the assembled replisome recapitulates all the key features of more complex prokaryotic and eukaryotic systems. In this review, we describe chemical mechanisms employed by individual proteins at the replication fork. Integration of structural, biochemical, and single-molecule data reveals a compelling view on how a nearly 1-MDa molecular machine acts as a unit to synthetize the two antiparallel DNA strands in a coordinated fashion.

Increased perinatal remodelling of the pancreas in somatostatin-deficient mice: potential role of transforming growth factor-beta signalling in regulating beta cell growth in early life.

Early postnatal life is a critical period for development of the endocrine pancreas, involving remodelling of islet cells and maturation of secretory responses. Factors that regulate these processes are undefined. Somatostatin-secreting delta-cells are abundant in the developing pancreas and, because somatostatin inhibits growth, the hormone may regulate islet expansion in early life. The aim of this study was to investigate effects of somatostatin-deficiency on proliferation, apoptosis and pancreas expansion in the first 3 weeks of life in mice. Pancreases from control or somatostatin-knockout mice were analysed for beta cell, alpha cell and pancreatic volumes by morphometry, proliferation by BrdU incorporation and apoptosis by TUNEL labelling. Signalling pathways associated with proliferation and apoptosis were studied by immunohistochemistry and Western blotting. Knockout mice grew normally in the first 3 weeks of life, but had high circulating insulin that normalised by day 21. Beta cell, alpha cell and pancreatic volumes were decreased in knockout mice, accompanied by reduced proliferation and increased apoptosis in the pancreas. Decreased growth was not due to impaired Akt signalling, as Akt phosphorylation and nuclear cyclin-D2 increased in the knockout pancreas. Levels of TGF-ß1, a factor implicated in tissue remodelling, together with SMAD phosphorylation through which TGF-ß mediates its effects, were increased in the knockout pancreas. Beta cell expansion was impaired in knockout mice, potentially compensating for increased insulin secretion from islets lacking inhibitory effects of somatostatin, and was associated with increased TGF-ß1 levels. TGF-ß1 may represent an important regulator of beta cell mass in early life.

Islets in early life are resistant to detrimental effects of a high-fat maternal diet: a study in rats.

Offspring of rats fed high-fat diets during pregnancy and lactation develop glucose intolerance and islet dysfunction in adulthood. Because other models of developmental programming of glucose intolerance are associated with defective islet development, we investigated whether high-fat exposure during fetal or neonatal life impairs islet development and function, thereby contributing to islet dysfunction in later life. Female rats were fed control or high-fat diets and their pups cross-fostered after birth to represent 4 groups with each combination of control and high-fat diet for the natural and foster mother. In a time course study, pups were kept with the natural mother until weaning. Pancreases were analysed for insulin content, beta cell mass, and islet number. Isolated islets were studied for insulin secretory responses and susceptibility to palmitate-induced apoptosis assessed by caspases 3/9 activity. Pancreatic insulin content and beta cell mass were increased in pups exposed to maternal high-fat diets after birth, whereas glucose-stimulated insulin secretion from islets of high-fat offspring at 5 and 11 days of age was lower than controls. Islets from control rats of 2-14 days of age were resistant to the pro-apoptotic effects of palmitate seen in older animals. The immature beta cell is therefore insensitive to toxic effects of palmitate and may compensate for the inhibitory effects on insulin secretion by increasing beta cell mass. The data suggest that susceptibility to glucose intolerance in offspring of dams fed high-fat diets may not be a consequence of deleterious effects on beta cell mass in early life.

Low levels of glucose transporters and K+ATP channels in human pancreatic beta cells early in development.

AIMS/HYPOTHESIS: Although cells expressing insulin are detected early in human fetal development, islets isolated from fetal pancreases show poor insulin secretory responses to glucose, which may be the result of deficient glucose sensing. We have used dual and triple immunolabelling of human fetal and adult pancreas sections to investigate the presence of proteins that participate in glucose sensing in the pancreatic beta cell, namely glucose transporter 1 (GLUT 1, also known as SLC2A1), glucose transporter 2 (GLUT2, also known as SLC2A2), glucokinase (GCK) and inwardly rectifying K+ channel (KIR6.2, also known as KCNJ11) and sulphonylurea receptor 1 (SUR1, also known as ABCC8) subunits of ATP-sensitive K+ channels (K+(ATP) channels). MATERIALS AND METHODS: Pancreases obtained with ethical approval from human fetuses from 11 to 36 weeks of gestation, from infants and from adults were formalin-fixed and embedded in paraffin. Sections were labelled with antibodies to proteins of interest. Co-production of antigens was examined by dual and triple immunolabelling. RESULTS: GLUT2 and K+(ATP) channel labelling was detected in the 11-week pancreas, but largely within the pancreatic epithelium, whereas no labelling for GLUT1 was observed. From 15 weeks, GLUT1, GCK and K+(ATP) channel labelling was detected in an increasing proportion of insulin-positive cells and epithelial labelling with K+(ATP) channel antibodies diminished. GLUT2 was seen in the majority of beta cells only after 7 months of age. CONCLUSIONS/INTERPRETATION: The results demonstrate that only a subpopulation of beta cells in the human fetal pancreas produce all key elements of the glucose-sensing apparatus, which may contribute to poor secretory responses in early life.

A role for kisspeptin in islet function.

AIMS/HYPOTHESIS: We investigated the production of kisspeptin (KISS1) and the KISS1 receptor, GPR54, in pancreatic islets and determined the effects of exogenous kisspeptin on insulin secretion. METHODS: RT-PCR and immunohistochemistry were used to detect expression of KISS1 and GPR54 mRNAs and the production of KISS1 and GPR54 in human and mouse islets and in beta (MIN6) and alpha- (alphaTC1) cell lines. The effects of KISS1 on basal and glucose-induced insulin secretion from mouse and human islets were measured in a perifusion system. RESULTS: KISS1 and GPR54 mRNAs were both detected in human and mouse islets, and GPR54 mRNA expression was also found in the MIN6 and alphaTC1 endocrine cell lines. In sections of mouse pancreas, KISS1 and GPR54 immunoreactivities were co-localised in both beta and alpha cells within islets, but were not detected in the exocrine pancreas. Exposure of mouse and human islets to KISS1 caused a stimulation of glucose-induced (20 mmol/l) insulin secretion, but had no effect on the basal rate of secretion at a sub-stimulatory concentration of glucose (2 mmol/l). In contrast, KISS1 inhibited insulin secretion from MIN6 cells at both 2 and 20 mmol/l glucose. KISS1 had no significant effect on glucagon secretion from mouse islets. CONCLUSIONS/INTERPRETATION: This is the first report to show that the GPR54/KISS1 system is expressed in the endocrine pancreas, where it influences beta cell secretory function. These observations suggest an important role for this system in the normal regulation of islet function.

Genomic analysis of bacteriophages SP6 and K1-5, an estranged subgroup of the T7 supergroup.

We have determined the genome sequences of two closely related lytic bacteriophages, SP6 and K1-5, which infect Salmonella typhimurium LT2 and Escherichia coli serotypes K1 and K5, respectively. The genome organization of these phages is almost identical with the notable exception of the tail fiber genes that confer the different host specificities. The two phages have diverged extensively at the nucleotide level but they are still more closely related to each other than either is to any other phage currently characterized. The SP6 and K1-5 genomes contain, respectively, 43,769 bp and 44,385 bp, with 174 bp and 234 bp direct terminal repeats. About half of the 105 putative open reading frames in the two genomes combined show no significant similarity to database proteins with a known or predicted function that is obviously beneficial for growth of a bacteriophage. The overall genome organization of SP6 and K1-5 is comparable to that of the T7 group of phages, although the specific order of genes coding for DNA metabolism functions has not been conserved. Low levels of nucleotide similarity between genomes in the T7 and SP6 groups suggest that they diverged a long time ago but, on the basis of this conservation of genome organization, they are expected to have retained similar developmental strategies.

Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.

At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C- and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5'-NNGTC-3' via a zinc motif and catalyzes the template-directed synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5'-ACCC-3' at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.

A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive.

Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for phage replication. A single point mutation in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain. Upon infection of Escherichia coli by the temperature-sensitive phage at 42 degrees C, there is no detectable T7 DNA synthesis in vivo. DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 degrees C or 42 degrees C. Upon infection at 30 degrees C, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degrees C or 42 degrees C approach levels observed using wild-type T7 phage. The amount of soluble gene 5 protein produced at 42 degrees C is comparable to that produced at 30 degrees C, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 30 degrees C closely resemble that of wild-type T7 DNA polymerase. It is likely that the polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway.

Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7.

The gene 2.5 protein (gp2.5) of bacteriophage T7 is a single-stranded DNA (ssDNA) binding protein that has essential roles in DNA replication and recombination. In addition to binding DNA, gp2.5 physically interacts with T7 DNA polymerase and T7 primase-helicase during replication to coordinate events at the replication fork. We have determined a 1.9-A crystal structure of gp2.5 and show that it has a conserved OB-fold (oligosaccharide/oligonucleotide binding fold) that is well adapted for interactions with ssDNA. Superposition of the OB-folds of gp2.5 and other ssDNA binding proteins reveals a conserved patch of aromatic residues that stack against the bases of ssDNA in the other crystal structures, suggesting that gp2.5 binds to ssDNA in a similar manner. An acidic C-terminal extension of the gp2.5 protein, which is required for dimer formation and for interactions with the T7 DNA polymerase and the primase-helicase, appears to be flexible and may act as a switch that modulates the DNA binding affinity of gp2.5.

Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.

The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.

DNA primases.

DNA primases are enzymes whose continual activity is required at the DNA replication fork. They catalyze the synthesis of short RNA molecules used as primers for DNA polymerases. Primers are synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long. Most DNA primases can be divided into two classes. The first class contains bacterial and bacteriophage enzymes found associated with replicative DNA helicases. These prokaryotic primases contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either is itself a DNA helicase or interacts with a DNA helicase. The second major primase class comprises heterodimeric eukaryotic primases that form a complex with DNA polymerase alpha and its accessory B subunit. The small eukaryotic primase subunit contains the active site for RNA synthesis, and its activity correlates with DNA replication during the cell cycle.

A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization.

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.

Spontaneous fracture (sfx): a mouse genetic model of defective peripubertal bone formation.

A new mouse model of stage-specific bone growth failure and fracture has been recovered as an autosomal recessive mutation, designated spontaneous fracture (sfx). The sfx/sfx mice are phenotypically normal until shortly after weaning, when reduced mobility and impaired somatic growth are first noted. By 6 weeks of age, body, spleen, and thymus weights, as well as hematocrits and serum calcium, inorganic phosphate, total alkaline phosphatase, insulin-like growth factor-I, and osteocalcin levels are decreased. The sfx/sfx mice also show reduced femoral cortical density and diaphyseal circumference, as well as a paucity of mature osteoblasts on bone surfaces. Histological analyses of the femur and tibia in the mutants show subtle reduction of chondrocyte numbers in epiphyseal-plate columns, reduction of matrix, and near absence of osteoid below the differentiated chondrocytes. Trabeculae in proximal tibiae, iliacs, and vertebral bodies are sparse and thin. Cortical bone thickness of mutants is markedly thinned in all sites examined. By 7-8 weeks, radiographic films routinely show spontaneous impact fractures of the distal femur accompanied by callus formation, whereas complete fractures are less commonly observed. Volumetric bone mineral density (BMD) of mutant femurs is similar to +/? littermates in the center of the femoral diaphysis, but BMD declines as either end of the femoral diaphysis is approached. We have mapped the gene responsible for this phenotype to central Chromosome 14. Reduced bone mass, impaired bone formation, abnormalities of bone architecture, and a disposition to spontaneous fracture identify sfx/sfx mice as a useful model for understanding the mechanisms responsible for peripubertal bone formation.

A unique loop in the DNA-binding crevice of bacteriophage T7 DNA polymerase influences primer utilization.

The three-dimensional structure of bacteriophage T7 DNA polymerase reveals the presence of a loop of 4 aa (residues 401-404) within the DNA-binding groove; this loop is not present in other members of the DNA polymerase I family. A genetically altered T7 DNA polymerase, T7 polDelta401-404, lacking these residues, has been characterized biochemically. The polymerase activity of T7 polDelta401-404 on primed M13 single-stranded DNA template is one-third of the wild-type enzyme and has a 3'-to-5' exonuclease activity indistinguishable from that of wild-type T7 DNA polymerase. T7 polDelta401-404 polymerizes nucleotides processively on a primed M13 single-stranded DNA template. T7 DNA polymerase cannot initiate de novo DNA synthesis; it requires tetraribonucleotides synthesized by the primase activity of the T7 gene 4 protein to serve as primers. T7 primase-dependent DNA synthesis on single-stranded DNA is 3- to 6-fold less with T7 polDelta401-404 compared with the wild-type enzyme. Furthermore, the altered polymerase is defective (10-fold) in its ability to use preformed tetraribonucleotides to initiate DNA synthesis in the presence of gene 4 protein. The location of the loop places it in precisely the position to interact with the tetraribonucleotide primer and, presumably, with the T7 gene 4 primase. Gene 4 protein also provides helicase activity for the replication of duplex DNA. T7 polDelta401-404 and T7 gene 4 protein catalyze strand-displacement DNA synthesis at nearly the same rate as does wild-type polymerase and T7 gene 4 protein, suggesting that the coupling of helicase and polymerase activities is unaffected.

Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6.

The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3'. This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. colibacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli- or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.

The motif D loop of human immunodeficiency virus type 1 reverse transcriptase is critical for nucleoside 5'-triphosphate selectivity.

Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the "palm" domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment from Taq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3'-deoxy 3'-azidothymidine 5'-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.

Interaction of bacteriophage T7 gene 4 primase with its template recognition site.

The primase fragment of the bacteriophage T7 63-kDa gene 4 helicase/primase protein contains the 271 N-terminal amino acid residues and lacks helicase activity. The primase fragment catalyzes the synthesis of oligoribonucleotides at rates similar to those catalyzed by the full-length protein in the presence of a 5-nucleotide DNA template containing a primase recognition site (5'-GGGTC-3', 5'-TGGTC-3', 5'-GTGTC-3', or 5'-TTGTC-3'). Although it is not copied into the oligoribonucleotides, the cytosine at the 3'-position is essential for synthesis and template binding. Two nucleotides flanking the 3'-end of the recognition site are required for tight DNA binding and rapid oligoribonucleotide synthesis. Nucleotides added to the 5'-end have no effect on the rate of oligoribonucleotide synthesis or the affinity of the primase for DNA. The binding of either ATP or CTP significantly increases the affinity of the primase for its DNA template. DNA lacking a primase recognition site does not inhibit oligoribonucleotide synthesis, suggesting that the primase binds DNA in a sequence-specific manner. The affinity of the primase for templates is weak, ranging from 10 to 150 microM. The tight DNA binding (<1 microM) observed with the 63-kDa gene 4 protein occurs via interactions between DNA templates and the helicase domain.

Interaction of ribonucleoside triphosphates with the gene 4 primase of bacteriophage T7.

The primase fragment of bacteriophage T7 gene 4 protein catalyzes the synthesis of oligoribonucleotides in the presence of ATP, CTP, Mg(2+) (or Mn(2+)), and DNA containing a primase recognition site. During chain initiation, ATP binds with a K(m) of 0.32 mM, and CTP binds with a K(m) of 0.85 mM. Synthesis of the dinucleotides proceeds at a rate of 3.8/s. The dinucleotide either dissociates or is extended to a tetranucleotide. The primase preferentially inserts ribonucleotides forming Watson-Crick base pairs with the DNA template >200-fold more rapidly than other ribo- or deoxynucleotides. 3'-dCTP binds the primase with a similar affinity as CTP and is incorporated as a chain terminator at a rate (1)/(100) that of CTP. ATP analogues alpha,beta-methylene ATP, beta,gamma-methylene ATP, and beta,gamma-imido ATP are incorporated by the primase fragment at the 5'-ends of the oligoribonucleotides but not at the 3'-ends. A model is presented in which the primase fragment utilizes two nucleotide-binding sites, one for the initiating ATP and one for the nucleoside triphosphate which elongates the primer on the 3'-end. The initiation site binds ATP or oligoribonucleotides, whereas the elongation site binds ATP or CTP as directed by the template.

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.

Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7.

Helicases that unwind DNA at the replication fork are ring-shaped oligomeric enzymes that move along one strand of a DNA duplex and catalyze the displacement of the complementary strand in a reaction that is coupled to nucleotide hydrolysis. The helicase domain of the replicative helicase-primase protein from bacteriophage T7 crystallized as a helical filament that resembles the Escherichia coli RecA protein, an ATP-dependent DNA strand exchange factor. When viewed in projection along the helical axis of the crystals, six protomers of the T7 helicase domain resemble the hexameric rings seen in electron microscopic images of the intact T7 helicase-primase. Nucleotides bind at the interface between pairs of adjacent subunits where an arginine is near the gamma-phosphate of the nucleotide in trans. The bound nucleotide stabilizes the folded conformation of a DNA-binding motif located near the center of the ring. These and other observations suggest how conformational changes are coupled to DNA unwinding activity.