The T4 DNA polymerase accessory proteins form an ATP-dependent complex on a primer-template junction

The DNA polymerase holoenzyme of bacteriophage T4 contains, besides the DNA polymerase itself (the gene 43 protein), a complex of the protein products of T4 genes 44 and 62 (a DNA-dependent ATPase) and of gene 45. Together, the 44/62 and 45 proteins form an ATP-dependent "sliding clamp" that holds a moving DNA polymerase molecule at the 3' terminus of a growing DNA chain. We have used a unique DNA fragment that forms a short hairpin helix with a single-stranded 5' tail (a "primer-template junction") to map the binding sites for these polymerase accessory proteins by DNA footprinting techniques. In the absence of the DNA polymerase, the accessory proteins protect from DNase I cleavage 19-20 nucleotides just behind the 3' end of the primer strand and 27-28 nucleotides on the complementary portion of the template strand. Detection of this DNA-protein complex requires the 44/62 and 45 proteins plus the nonhydrolyzable ATP analogue adenosine 5'-O-(thiotriphosphate). The complex is not detected in the presence of ATP. We suggest that ATP hydrolysis by the 44/62 protein normally activates the accessory proteins at a primer-template junction, permitting the DNA polymerase to bind and thus form the complete holoenzyme. However, when the polymerase is missing, as in these experiments, ATP hydrolysis is instead followed by a release (or loosening) of the accessory protein complex.     

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.     

The complex of T4 bacteriophage gene 44 and 62 replication proteins forms an ATPase that is stimulated by DNA and by T4 gene 45 protein

The bacteriophage T4 genome is believed to encode all of the proteins needed for the replication of its own DNA. Included among these proteins are the "polymerase accessory proteins", the products of T4 genes 44, 62 and 45. The first two of these genes specify the synthesis of the 44/62 protein complex, which is here shown to be a DNA-dependent ATPase, hydrolyzing either ATP or dATP to the corresponding nucleoside diphosphate and releasing inorganic phosphate. This nucleotide hydrolysis is greatly stimulated by addition of the gene 45 protein and by single-stranded DNA termini. A rapid micro DNA-cellulose assay is introduced and used to measure accessory protein binding to the complex of T4 gene 32 protein and single-stranded DNA. In the presence of ATP, the 44/62 protein binds to this complex but not to naked DNA, while the 45 protein requires both the 32 protein and the 44/62 protein for detectable binding.     

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.     

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.     

Structural and enzymatic studies of the T4 DNA replication system. II. ATPase properties of the polymerase accessory protein complex

In this paper we report a detailed enzymatic characterization of the interaction of the polymerase accessory protein complex of the T4 DNA replication system with the various nucleic acid cofactors that activate the ATPase of the complex. We show that the ATPase activity of the T4 coded gene 44/62 protein complex is stimulated synergistically by binding of DNA and T4 gene 45 protein and that the level of ATPase activation appears to be directly correlated with the binding of nucleic acid cofactor. Binding of any partially or completely single-stranded DNA to the complete accessory protein complex increases the catalytic activity (as measured by Vmax) while decreasing the binding affinity for the ATP substrate. While single-stranded DNA is a moderately effective cofactor, we find that the optimal nucleic acid-binding site for the complex is the primer-template junction, rather than single-stranded DNA ends as previously reported in the literature. Gene 45 protein plays an essential role in directing the specificity of binding to primer-template sites, lowering the Km for primer-template sites almost 1000-fold, and increasing Vmax 100-fold, compared with the analogous values for gene 44/62 protein alone. The most effective primer-template site for binding and enzymatic activation has the physiologically relevant recessed 3'-OH configuration and an optimal size in excess of 18 base pairs of duplex DNA. We find that the chemical nature of the primer terminus (i.e. 3'-OH or 3'-H) does not affect the extent of ATPase activation and that binding of the polymerase accessory protein complex to DNA cofactors is salt concentration dependent but appreciably less so when the activating DNA is a primer-template junction. Finally, we show that the gene 32 protein (T4 coded single-stranded DNA-binding protein) can compete with the polymerase accessory protein complex for single-stranded DNA but not for the primer-template junction activation sites. The implications of these results for the structure and function of the polymerase accessory protein complex within the T4 DNA replication system are discussed.     

The 44P subunit of the T4 DNA polymerase accessory protein complex catalyzes ATP hydrolysis

The genes encoding all three T4 DNA polymerase accessory proteins have been cloned into overexpression plasmids. Induction of cells harboring these plasmids results in the synthesis of each accessory protein at levels that approach 10% of the total cellular protein. The solubility of the accessory proteins after induction at 42 degrees C ranges from about 60% to greater than 95%. A plasmid that allows overexpression of the 44P/62P complex has been manipulated further to overexpress selectively the 44P subunit without 62P, permitting us to assess how each subunit contributes to the properties of the 44P/62P complex. A comparison of 44P and 44P/62P by conventional hydrodynamic techniques shows that 44P forms a subcomplex nearly as large as the 44P/62P complex. In addition, 44P catalyzes DNA-dependent ATP hydrolysis with a specific activity similar to that of the 44P/62P ATPase. However, unlike the 44P/62P complex, the ATPase activity of 44P alone is only slightly stimulated by 45P. This suggests that one role of the 62P subunit is to facilitate a productive interaction of 44P and 45P.     

Characterization of the stimulatory effect of T4 gene 45 protein and the gene 4462 protein complex on DNA synthesis by T4 DNA polymerase

On a variety of single-stranded DNA templates, the overall rate of in vitro DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase is increased about fourfold by addition of the T4 gene $\text{44}\text{62}$ and 45 proteins. Several different methods suggest that this stimulation reflects an increase in the average DNA polymerase “sticking distance”, or processivity, from 800 to about 3000 nucleotides per initiation event. Both the $\text{44}\text{62}$ protein complex and the 45 protein must be present to obtain this effect, and either ATP or dATP hydrolysis is required. Rapid-mixing experiments indicate that the polymerase stimulation is maximized within a few seconds after addition of these “polymerase accessory proteins.”     

An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex

In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form in solution raises the possibility of other pathways for clamp loading. In this study, an alternative clamp loading sequence is evaluated in which gp44/62 first recognizes the DNA substrate and then sequesters the clamp from solution and loads it onto DNA. This pathway differs in terms of the initial formation of a gp44/62-DNA complex that is capable of loading gp45. In this work, we demonstrate ATP-dependent DNA binding by gp44/62. Among various DNA structures that were tested, gp44/62 binds specifically to primer-template DNA but not to single-stranded DNA or blunt-end duplex DNA. By tracing the dynamic clamp closing with pre-steady-state FRET measurements, we show that the clamp loader-DNA complex is functional in clamp loading. Furthermore, pre-steady-state ATP hydrolysis experiments suggest that 1 equiv of ATP is hydrolyzed when gp44/62 binds to DNA, and additional ATP hydrolysis is associated with the completion of the clamp loading process. We also investigated the detailed kinetics of binding of MANT-nucleotide to gp44/62 through stopped-flow FRET and demonstrated a conformational change as the result of ATP, but not ADP binding. The collective kinetic data allowed us to propose and evaluate a sequence of steps describing this alternative pathway for clamp loading and holoenzyme formation.     

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.     

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.     

Stimulation of the processivity of the DNA polymerase of bacteriophage T4 by the polymerase accessory proteins. The role of ATP hydrolysis

In this paper we examine the role of the DNA polymerase accessory proteins in modulating the processivity of DNA synthesis by the bacteriophage T4-coded five protein "holoenzyme" replication complex in vitro. Primed single-stranded DNA was used as a template for the DNA synthesis reactions, and buffer conditions were chosen to mimic in vivo salt concentrations. We find that the accessory proteins significantly increase the DNA-bound lifetime of the holoenzyme complex but that the maximum lifetime of the complex is still less than 10 s at 22 degrees C. The accessory proteins greatly enhance the processivity of the holoenzyme relative to that of the polymerase alone. ATP hydrolysis catalyzed by the accessory proteins complex is required to achieve this enhancement. We have investigated the temporal relationship between ATP hydrolysis by the accessory proteins and primer elongation by the holoenzyme and find that ATPase activity is required for initial assembly of the holoenzyme complex but not for elongation per se. Thus we conclude that the increased processivity displayed by the holoenzyme in moving through regions of template secondary structure reflects the high intrinsic processivity of the holoenzyme complex itself rather than a requirement for a concomitant ATPase-driven helicase activity during elongation. We have also measured the ATPase activity of the accessory proteins as a function of polymerase concentration and find that the rate of ATP hydrolysis catalyzed by this complex decreases significantly when the accessory proteins are assembled (with polymerase and gene 32 protein) into the five-protein holoenzyme and coupled to primer elongation. Based on these results we discuss mechanisms by which the ATPase activity of the polymerase accessory proteins might stimulate the overall processivity of the holoenzyme.     

Persistence of DNA synthesis arrest sites in the presence of T4 DNA polymerase and T4 gene 32, 44, 45 and 62 DNA polymerase accessory proteins.

DNA synthesis by phage T4 DNA polymerase is arrested at specific sequences in single-stranded DNA templates. To determine whether or not T4 DNA polymerase accessory proteins 32, 44, 45 and 62 eliminated recognition of these arrest sites, unique primer-templates were constructed in which DNA synthesis began at a DNA primer located at different distances from palindromic and nonpalindromic arrest sites. Nucleotide positions that caused polymerase to pause or leave the template were identified by sequence analysis of 5'-end labeled nascent DNA chains. Stable hairpin structures at palindromic sequences were confirmed by acetylation of single-stranded sequences with bromoacetaldehyde. Our results confirmed that these T4 DNA polymerase accessory proteins stimulated T4 DNA polymerase activity and processivity on natural as well as homopolymer primer-templates. However, they did not alter recognition of DNA synthesis arrest sites by T4 DNA polymerase. Extensive DNA synthesis resulted from an increased rate of translocation and/or processivity to the same extent over all DNA sequences.     

Processivity clamp gp45 and ssDNA-binding-protein gp32 modulate the fidelity of bacteriophage RB69 DNA polymerase in a sequence-specific manner, sometimes enhancing and sometimes compromising accuracy

Numerous studies of the impact of accessory proteins upon the fidelity of DNA synthesis have provided a complex and sometimes discordant picture. We previously described such an analysis conducted in vitro using various bacteriophage RB69 gp43 mutator DNA polymerases with or without the accessory proteins gp32 (which binds single-stranded DNA) plus gp45/44/62 (processivity clamp and its loaders). Mutations were scored at many sites in the lacZalpha mutation reporter sequence. Unexpectedly, the accessory proteins sometimes decreased and sometimes increased fidelity at a handful of specific sites. Here, we enlarge our analysis with one particular mutator polymerase compromised in both insertion accuracy and proofreading and also extend the analysis to reactions supplemented only with gp32 or only with gp45/44/62. An overall 1.56-fold increase in mutation frequencies was produced by adding single or multiple accessory proteins and was driven mainly by increased T(template)*G(primer) mispairs. Evidence was found for many additional sites where the accessory proteins influence fidelity, indicating the generality of the effect. Thus, accessory proteins contribute to the site-specific variability in mutation rates characteristically seen in mutational spectra.     

Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins.

Eukaryotic DNA polymerase delta and its accessory proteins are essential for SV40 DNA replication in vitro. A multi-subunit protein complex, replication factor C (RF-C), which is composed of subunits with apparent molecular weights of 140,000, 41,000, and 37,000, has primer/template binding and DNA-dependent ATPase activities. UV-cross-linking experiments demonstrated that the Mr = 140,000 subunit recognizes and binds to the primer-template DNA, whereas the Mr = 41,000 polypeptide binds ATP. Assembly of a replication complex at a primer-template junction has been studied in detail with synthetic, hairpin DNAs. Following glutaraldehyde fixation, a gel shift assay demonstrated that RF-C alone forms a weak binding complex with the hairpin DNA. Addition of ATP or its nonhydrolyzable analogue, ATP gamma S, increased specific binding to the DNA. Footprinting experiments revealed that RF-C recognizes the primer-template junction, covering 15 bases of the primer DNA from the 3'-end and 20 bases of the template DNA. Another replication factor, proliferating cell nuclear antigen (PCNA) binds to RF-C and the primer-template DNA forming a primer recognition complex and extends the protected region on the duplex DNA. This RF-C.PCNA complex has significant single-stranded DNA binding activity in addition to binding to a primer-template junction. However, addition of another replication factor, RF-A, completely blocked the nonspecific, single-stranded DNA binding by the RF-C.PCNA complex. RF-A therefore functions as a specificity factor for primer recognition. In the absence of RF-C, DNA polymerase delta (pol delta) and PCNA form a complex at the primer-template junction, protecting exactly the same site as the primer recognition complex. Addition of RF-C to this complex produced a higher order complex which is unstable unless its formation is coupled with translocation of pol delta. These results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin. We demonstrate this directly in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1961-1968).     

Conditional coupling of leading-strand and lagging-strand DNA synthesis at bacteriophage T4 replication forks

Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution. If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed. Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis. A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis. Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA. It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase. Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner.     

Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer and DNA synthesis

Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.     

Biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69

The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering. We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action. We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable. The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction.     

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.