An ATP stimulation of T4 DNA polymerase mediated via T4 gene 44/62 and 45 proteins. The requirement for ATP hydrolysis.

An in vitro replication system reconstituted from six purified T4 bacteriophage proteins, each of which is essential for T4 DNA replication in vivo, requires ATP. Because of the complexity of the complete system, we examine in this report the involvement of ATP in two subsystems of the overall DNA synthesis reaction. One subsystem consists of the T4 DNA polymerase (gene 43 protein) and its "accessory proteins," the gene 44/62 and 45 products. An even simpler subsystem consists of the gene 44/62 and 45 proteins alone, which together have a DNA-dependent ATPase activity. The combination of the 44/62 and 45 proteins hydrolyze ATP to ADP and inorganic phosphate in the presence of DNA. These essential accessory proteins have been previously shown to increase T4 DNA polymerase activity on primed, single-stranded DNA templates. In this report we use nucleotide analogues to demonstrate that this polymerase stimulation requires hydrolysis of the beta,gamma-phosphate bond of ATP. However, our data suggest that the mechanism of accessory protein stimulation is such that less than 1 ATP molecule need be hydrolyzed per 10 deoxyribonucleotides incorporated by the DNA polymerase into DNA.     

Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase

Bacteriophage T4 gene 41 protein is one of the two phage proteins previously shown to be required for the synthesis of the pentaribonucleotide primers which initiate the synthesis of new chains in the T4 DNA replication system. We now show that a DNA helicase activity which can unwind short fragments annealed to complementary single-stranded DNA copurifies with the gene 41 priming protein. T4 gene 41 is essential for both the priming and helicase activities, since both are absent after infection by T4 phage with an amber mutation in gene 41. A complete gene 41 product is also required for two other activities previously found in purified preparations of the priming activity: a single-stranded DNA-dependent GTPase (ATPase) and an activity which stimulates strand displacement synthesis catalyzed by T4 DNA polymerase, the T4 gene 44/62 and 45 polymerase accessory proteins, and the T4 gene 32 helix-destabilizing protein (five-protein reaction). The 41 protein helicase requires a single-stranded DNA region adjoining the duplex region and begins unwinding at the 3' terminus of the fragment. There is a sigmoidal dependence on both nucleotide (rGTP, rATP) and protein concentration for this reaction. 41 Protein helicase activity is stimulated by our purest preparation of the T4 gene 61 priming protein, and by the T4 gene 44/62 and 45 polymerase accessory proteins. The direction of unwinding is consistent with the idea that 41 protein facilitates DNA synthesis on duplex templates by destabilizing the helix as it moves 5' to 3' on the displaced strand.     

DNA replication by bacteriophage T4 proteins. The T4 43, 32, 44--62, And 45 proteins are required for strand displacement synthesis at nicks in duplex DNA.

The T4 bacteriophage gene 43 (T4 DNA polymerase), 32 (DNA helix-destabilizing protein), and 45 proteins and the complex of the gene 44 and 62 proteins are all required for DNA synthesis beginning at single-stranded breaks in duplex DNA. This synthesis occurs by strand displacement and is not dependent on ribonucleotides, the T4 gene 41 protein, or the T4 initiating protein, each of which is required to begin new chains on single-stranded templates. Electron microscopic analysis shows that duplex molecules with long single-stranded branches are the predominant products of this strand displacement synthesis.     

RNA priming of DNA replication by bacteriophage T4 proteins

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.     

Trypsin cleavage in the COOH terminus of the bacteriophage T4 gene 41 DNA helicase alters the primase-helicase activities of the T4 replication complex in vitro

The bacteriophage T4 gene 41 protein is a 5' to 3' DNA helicase which unwinds DNA ahead of the growing replication fork and, together with the T4 gene 61 protein, also functions as a primase to initiate DNA synthesis on the lagging strand. Proteolytic cleavage by trypsin approximately 20 amino acids from the COOH terminus of the 41 protein produces 41T, a 51,500-dalton fragment (possibly still associated with small COOH-terminal fragments) which still retains the ssDNA-stimulated GTPase (ATPase) activity, the 61 protein-stimulated DNA helicase activity, and the ability to act with 61 protein to synthesize pentaribonucleotide primers. In the absence of the T4 gene 32 ssDNA binding protein, the primase-helicase composed of the tryptic fragment (41T) and 61 proteins efficiently primes DNA synthesis on circular ssDNA templates by the T4 DNA polymerase and the three T4 polymerase accessory proteins. In contrast, the 41T protein is defective as a helicase or a primase component on 32 protein-covered DNA. Thus, unlike the intact protein, 41T does not support RNA-dependent DNA synthesis on 32 protein-covered ssDNA and does not stimulate strand displacement DNA synthesis on a nicked duplex DNA template. High concentrations of 32 protein strongly inhibit RNA primer synthesis with either 41 T or intact 41 protein. The 44/62 and 45 polymerase accessory proteins (and even the 44/62 proteins to some extent) substantially reverse the 32 protein inhibition of RNA primer synthesis with intact 41 protein but not with 41T protein. We propose that the COOH-terminal region of the 41 protein is required for its interaction with the T4 polymerase accessory proteins, permitting the synthesis and utilization of RNA primers and helicase function within the T4 replication complex. When this region is altered, as in 41T protein, the protein is unable to assemble a functional primase-helicase in the replication complex. An easy and rapid purification of T4 41 protein produced by a plasmid encoding this gene (Hinton, D. M., Silver, L. L., and Nossal, N. G. (1985) J. Biol. Chem. 260, 12851-12857) is also described.     

DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction

We have used DNA footprinting techniques to analyze the interactions of five DNA replication proteins at a primer-template junction: the bacteriophage T4 DNA polymerase (the gene 43 protein), its three accessory proteins (the gene 44/62 and 45 proteins), and the gene 32 protein, which is the T4 helix-destabilizing (or single-stranded DNA-binding) protein. The 177-nucleotide-long DNA substrate consisted of a perfect 52-base pair hairpin helix with a protruding single-stranded 5' tail. As expected, the DNA polymerase binds near the 3' end of this molecule (at the primer-template junction) and protects the adjacent double-stranded region from cleavage. When the gene 32 protein binds to the single-stranded tail, it reduces the concentration of the DNA polymerase required to observe the polymerase footprint by 10-30-fold. Periodic ATP hydrolysis by the 44/62 protein is required to maintain the activity of the DNA polymerase holoenzyme (a complex of the 43, 44/62, and 45 proteins). Footprinting experiments demonstrate the formation of a weak complex between the DNA polymerase and the gene 45 protein, but there is no effect of the 44/62 protein or ATP on this enlarged footprint. We propose a model for holoenzyme function in which the complex of the three accessory proteins uses ATP hydrolysis to keep a moving polymerase tightly bound to the growing 3' end, providing a "clock" to measure polymerase stalling.     

Prokaryotic DNA replication mechanisms

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.     

Function and assembly of the bacteriophage T4 DNA replication complex: interactions of the T4 polymerase with various model DNA constructs

Complexes formed between DNA polymerase and genomic DNA at the replication fork are key elements of the replication machinery. We used sedimentation velocity, fluorescence anisotropy, and surface plasmon resonance to measure the binding interactions between bacteriophage T4 DNA polymerase (gp43) and various model DNA constructs. These results provide quantitative insight into how this replication polymerase performs template-directed 5' --> 3' DNA synthesis and how this function is coordinated with the activities of the other proteins of the replication complex. We find that short (single- and double-stranded) DNA molecules bind a single gp43 polymerase in a nonspecific (overlap) binding mode with moderate affinity (Kd approximately 150 nm) and a binding site size of approximately 10 nucleotides for single-stranded DNA and approximately 13 bp for double-stranded DNA. In contrast, gp43 binds in a site-specific (nonoverlap) mode and significantly more tightly (Kd approximately 5 nm) to DNA constructs carrying a primer-template junction, with the polymerase covering approximately 5 nucleotides downstream and approximately 6-7 bp upstream of the 3'-primer terminus. The rate of this specific binding interaction is close to diffusion-controlled. The affinity of gp43 for the primer-template junction is modulated specifically by dNTP substrates, with the next "correct" dNTP strengthening the interaction and an incorrect dNTP weakening the observed binding. These results are discussed in terms of the individual steps of the polymerase-catalyzed single nucleotide addition cycle and the replication complex assembly process. We suggest that changes in the kinetics and thermodynamics of these steps by auxiliary replication proteins constitute a basic mechanism for protein coupling within the replication complex.     

Effects of the bacteriophage T4 dda protein on DNA synthesis catalyzed by purified T4 replication proteins

The T4 bacteriophage dda protein is a DNA-dependent ATPase and DNA helicase that is the product of an apparently nonessential T4 gene. We have examined its effects on in vitro DNA synthesis catalyzed by a purified, multienzyme T4 DNA replication system. When DNA synthesis is catalyzed by the T4 DNA polymerase on a single-stranded DNA template, the addition of the dda protein is without effect whether or not other replication proteins are present. In contrast, on a double-stranded DNA template, where a mixture of the DNA polymerase, its accessory proteins, and the gene 32 protein is required, the dda protein greatly stimulates DNA synthesis. The dda protein exerts this effect by speeding up the rate of replication fork movement; in this respect, it acts identically with the other DNA helicase in the T4 replication system, the T4 gene 41 protein. However, whereas a 41 protein molecule remains bound to the same replication fork for a prolonged period, the dda protein seems to be continually dissociating from the replication fork and rebinding to it as the fork moves. Some gene 32 protein is required to observe DNA synthesis on a double-stranded DNA template, even in the presence of the dda protein. However, there is a direct competition between this helix-destabilizing protein and the dda protein for binding to single-stranded DNA, causing the rate of replication fork movement to decrease at a high ratio of gene 32 protein to dda protein. As shown elsewhere, the dda protein becomes absolutely required for in vitro DNA synthesis when E. coli RNA polymerase molecules are bound to the DNA template, because these molecules otherwise stop fork movement (Bedinger, P., Hochstrasser, M., Jongeneel, C.V., and Alberts, B. M. (1983) Cell 34, 115-123).     

Purification of the T4 gene 32 protein free from detectable deoxyribonuclease activities.

Detailed procedures are presented which allow reproducible preparation of T4 gene 32 protein, a helix-destabilizing protein essential for DNA replication and genetic recombination in T4 bacteriophage-infected Escherichia coli cells. Although 32 protein can be purified to better than 99% homogeneity by any one of several procedures, these methods have been developed to remove trace amounts of contaminating deoxyribonucleases, which are present in high levels in the original infected cells. Two alternative preparations are presented, each involving three chromatographic steps. Both 32 proteins obtained are essentially "nuclease-free," when tested at physiological salt concentrations. However, we show here that the phenyl-Sepharose chromatography step, which is necessary to remove an exonuclease activity active only at low salt concentrations, also removes a second protein present in trace amounts. In some cases, retention of this second protein is desirable, since it is essential for obtaining RNA primed, de novo DNA chain starts in an in vitro DNA replication system, when this system is constructed by mixing highly purified preparations of each of the six replication proteins coded for by T4 genes 32, 43, 44, 62, 45, and 41.     

Two forms of the DNA polymerase of bacteriophage T7

The DNA polymerase induced by bacteriophage T7 can be isolated in two different forms. The distinguishing properties are: 1) the specific activities of the associated 3' to 5' single- and double-stranded DNA exonuclease activities, 2) the ability to catalyze DNA synthesis and strand displacement at nicks, and 3) the degree of stimulation of DNA synthesis on nicked, duplex DNAs by the gene 4 protein of phage T7. Form I is obtained when purification is carried out in the absence of EDTA while Form II is obtained if all purification steps are carried out in the presence of 0.1 mM EDTA. Form I has low levels of both exonuclease activities, less than 5% of those of Form II. Form I can initiate DNA synthesis at nicks leading to strand displacement, a consequence of which is its ability to be stimulated manyfold by the helicase activity of gene 4 protein on nicked, duplex templates. On the other hand, Form II cannot initiate synthesis at nicks even in the presence of gene 4 protein. In keeping with its higher exonuclease activities, Form II of T7 DNA polymerase has higher turnover of nucleotides activity (5-fold higher than Form I) and exhibits greater fidelity of nucleotide incorporation, as indicated by the rate of incorporation of 2-aminopurine deoxynucleoside monophosphate. Both forms of T7 DNA polymerase exhibit higher fidelity of nucleotide incorporation than bacteriophage T4 DNA polymerase. In the absence of EDTA or in the presence of FeSO4 or CaCl2, Form II irreversibly converts to Form I. The physical difference between the two forms is not known. No difference in molecular weight can be detected between the corresponding subunits of each form of T7 DNA polymerase as measured by gel electrophoresis in the presence of sodium dodecyl sulfate.     

Proteolytic removal of the COOH terminus of the T4 gene 32 helix-destabilizing protein alters the T4 in vitro replication complex

The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. 1. Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32I protein for this synthesis. 2. Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. 3. Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. 4. The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3'-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities.     

Formation of rolling-circle molecules during phi X174 complementary strand DNA replication

The primosome is a mobile multiprotein priming apparatus that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC and dnaG genes; replication factor Y (protein n'); and proteins i, n, and n"). While the primosome is analagous to the phage T7 gene 4 protein and phage T4 gene 41/61 proteins in its DNA G-catalyzed priming function, its ability to act similarly also as a DNA helicase has remained equivocal. The role of the primosome in unwinding duplex DNA strands was investigated in the coliphage phi X174 SS(c)----replicative form DNA replication reaction in vitro, which requires the E. coli single-stranded DNA binding protein, the primosomal proteins, and the DNA polymerase III holoenzyme. Multigenome-length, linear, double-stranded DNA molecules were generated in this reaction, presumably via a rolling circle-type mechanism. Synthesis of these products required the presence of a helicase-catalyzed strand-displacement activity to permit multiple cycles of continuous complementary (-) strand synthesis. The participation of the primosome in this helicase activity was supported by demonstrating that other SS(c) DNA templates (G4 and alpha-3), which lack primosome assembly sites, failed to support significant linear multimer production and that replication of phi X174 with the general priming system (the DNA B and DNA G proteins and DNA polymerase III holoenzyme) resulted in a 13-fold lower rate of linear multimer synthesis.     

Bacteriophage T4 DNA replication protein 41. Cloning of the gene and purification of the expressed protein

The bacteriophage T4 primase, composed of the T4 proteins 41 and 61, synthesizes pentaribonucleotides used to prime DNA synthesis on single-stranded DNA in vitro. 41 protein is also a DNA helicase that opens DNA in the same direction as the growing replication fork. Previously, Mattson et al. (Mattson, T., Van Houwe, G., Bolle, A., Selzer, G., and Epstein, R. (1977) Mol. Gen. Genet. 154, 319-326) located part of gene 41 on a 3400-base pair EcoRI fragment of T4 DNA (map units 24.3 to 21.15). In this paper, we report the cloning of T4 DNA representing map units 24.3 to 20.06 in a multicopy plasmid vector. Extracts of cells containing this plasmid complement gene 41- extracts in a DNA synthesis assay, indicating that this region contains all the information necessary for the expression of active 41 protein. We located gene 41 more precisely between T4 map units 22.01 to 20.06 since our cloning of this region downstream of the strong lambda promoter PL results in the production of active 41 protein at a level 100-fold greater than after T4 infection. We have purified 133 mg of homogeneous 41 protein from 27 g of these cells. Like the 41 protein from T4 infected cells, the purified 41 protein in conjunction with the T4 gene 61 priming protein catalyzes primer formation (assayed by RNA primer-dependent DNA synthesis with T4 polymerase, the genes 44/62 and 45 polymerase accessory proteins, and the gene 32 helix-destabilizing protein) and is a helicase whose activity is stimulated by T4 61 protein.     

The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7

In this paper we compare the effect of single-stranded DNA-binding proteins of bacteriophage T7 (gene 2.5 protein) and of Escherichia coli (SSB) at the T7 replication fork. The T7 gene 4 protein acts processively as helicase to promote leading strand synthesis and distributively as primase to initiate lagging strand synthesis by T7 DNA polymerase. On a nicked double-stranded template, the formation of a replication fork requires partial strand displacement so that gene 4 protein may bind to the displaced strand and unwind the helix catalytically. Both the T7 gene 2.5 protein and E. coli SSB act stoichiometrically to promote this initial strand displacement step. Once initiated, processive leading strand synthesis is not greatly stimulated by the single-stranded DNA-binding proteins. However, the T7 gene 2.5 protein, but not E. coli SSB, increases the frequency of initiation of lagging strand synthesis by greater than 10-fold. The results suggest a specific interaction of the T7 gene 2.5 protein with the T7 replication apparatus.     

Gene 4 protein of bacteriophage T7. Purification physical properties, and stimulation of T7 DNA polymerase during the elongation of polynucleotide chains.

With the use of an in vitro complementation assay to measure activity, the gene 4 protein of bacteriophage T7 has been purified 1000-fold to yield a nearly homogeneous protein. The purified gene 4 protein is a single polypeptide having a molecular weight of 58,000. In addition to being essential for T7 DNA replication in vivo and in vitro, the gene 4 protein is required for DNA synthesis by the purified T7 DNA polymerase on duplex T7 DNA templates. In the absence of ribonucleoside 5'-triphosphates, DNA synthesis by the gene 4 protein and the T7 DNA polymerase is dependent on phosphodiester bond interruptions containing 3'-hydroxyl groups (nicks) in the duplex DNA. The reaction is specific for the T7 DNA polymerase, but any duplex DNA containing nicks can serve as template. The Km for nicks in the reaction is 3 x 10(-10) M.     

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.     

Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model.

The DNA replication system of bacteriophage T4 serves as a relatively simple model for the types of reactions and protein-protein interactions needed to carry out and coordinate the synthesis of the leading and lagging strands of a DNA replication fork. At least 10 phage-encoded proteins are required for this synthesis: T4 DNA polymerase, the genes 44/62 and 45 polymerase accessory proteins, gene 32 single-stranded DNA binding protein, the genes 61, 41, and 59 primase-helicase, RNase H, and DNA ligase. Assembly of the polymerase and the accessory proteins on the primed template is a stepwise process that requires ATP hydrolysis and is strongly stimulated by 32 protein. The 41 protein helicase is essential to unwind the duplex ahead of polymerase on the leading strand, and to interact with the 61 protein to synthesize the RNA primers that initiate each discontinuous fragment on the lagging strand. An interaction between the 44/62 and 45 polymerase accessory proteins and the primase-helicase is required for primer synthesis on 32 protein-covered DNA. Thus it is possible that the signal for the initiation of a new fragment by the primase-helicase is the release of the polymerase accessory proteins from the completed adjacent fragment.     

Deoxyribonucleic acid polymerase of bacteriophage T7. Purification and properties of the phage-encoded subunit, the gene 5 protein.

DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-specified thioredoxin. The gene 5 protein has been purified 7400-fold to homogeneity from bacteriophage T7-infected Escherichia coli 7400 trxA cells that lack thioredoxin. The purification procedure has been monitored by using a complementation assay in which thioredoxin interacts with the gene 5 protein to form an active DNA polymerase. The purified gene 5 protein is a single polypeptide having a molecular weight of 87,000. The gene 5 protein itself has only 1 to 2% of the polymerase activity of T7 DNA polymerase. However, T7 DNA polymerase can be reconstituted by the addition of homogeneous thioredoxin to the gene 5 protein. Optimal reconstitution is obtained when the molar ratio of thioredoxin/gene 5 protein is 150. Under these conditions, the gene 5 protein attains approximately 80% of the activity of an equal amount of T7 DNA polymerase. The apparent Km for thioredoxin in the reaction to restore DNA polymerase activity is 2.8 x 10(-8) M. The enzymatic properties of the reconstituted enzyme are indistinguishable from those of T7 DNA polymerase synthesized in vivo; the reconstituted polymerase interacts with T7 gene 4 protein to catalyze DNA synthesis on duplex DNA templates.