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.     

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.     

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.     

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.     

Bacteriophage T4 gene 44 DNA polymerase accessory protein. Sequences of gene 44 and its protein product

Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication. We have isolated the gene for 44 protein from a previously constructed lambda-T4 hybrid phage (Wilson, G. G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 156, 203-214). We report here the nucleotide sequence of gene 44 and about 60 nucleotides 5' upstream from its coding region, which is immediately adjacent to gene 45. We have also purified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization. Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencing of peptides or by matching amino acid compositions of purified peptides. A total of 84% of the predicted amino acids was confirmed by the protein data. These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated Mr = 35,371. The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes. In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.     

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.     

RNA primer handoff in bacteriophage T4 DNA replication: the role of single-stranded DNA-binding protein and polymerase accessory proteins

In T4 phage, coordinated leading and lagging strand DNA synthesis is carried out by an eight-protein complex termed the replisome. The control of lagging strand DNA synthesis depends on a highly dynamic replisome with several proteins entering and leaving during DNA replication. Here we examine the role of single-stranded binding protein (gp32) in the repetitive cycles of lagging strand synthesis. Removal of the protein-interacting domain of gp32 results in a reduction in the number of primers synthesized and in the efficiency of primer transfer to the polymerase. We find that the primase protein is moderately processive, and this processivity depends on the presence of full-length gp32 at the replication fork. Surprisingly, we find that an increase in the efficiency of primer transfer to the clamp protein correlates with a decrease in the dissociation rate of the primase from the replisome. These findings result in a revised model of lagging strand DNA synthesis where the primase remains as part of the replisome after each successful cycle of Okazaki fragment synthesis. A delay in primer transfer results in an increased probability of the primase dissociating from the replication fork. The interplay between gp32, primase, clamp, and clamp loader dictates the rate and efficiency of primer synthesis, polymerase recycling, and primer transfer to the polymerase.     

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.     

Structural and enzymatic studies of the T4 DNA replication system. I. Physical characterization of the polymerase accessory protein complex

In this study, we have investigated the structural and physical properties of the bacteriophage T4 DNA polymerase accessory proteins. We find that T4 gene 44 and 62 proteins associate to form a tight, highly homogeneous complex, containing four gene 44 protein subunits and one gene 62 protein subunit. The molecular mass of the complex is 163,700 daltons. Sedimentation results suggest that the complex is quite asymmetric, with a prolate ellipsoid axial ratio of about 5:1. This protein complex is known to carry a DNA-dependent ATPase activity; we show by photoaffinity labeling that the ATP-binding sites reside in the gene 44 protein subunits of the complex. Equilibrium sedimentation and chemical cross-linking studies indicate that the T4 gene 45 protein self-associates to form a trimer in solution. This trimer species also appears to be quite asymmetric, showing an axial ratio for a prolate ellipsoid of about 6:1, assuming normal hydration.     

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.     

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.     

Translesion replication by DNA polymerase delta depends on processivity accessory proteins and differs in specificity from DNA polymerase beta

Mutations caused by DNA damage lead to the development of cancer. The critical step in the formation of these mutations is the replication of unrepaired lesions in DNA by DNA polymerases, a process termed translesion replication. Using a newly developed method for preparation of gapped plasmids, containing a site-specific synthetic abasic site, we analyzed translesion replication with purified mammalian DNA polymerases delta and beta. DNA polymerase delta was found to be unable to replicate through the abasic site. Addition of the sliding DNA clamp PCNA, the clamp loader RFC, and ATP caused a drastic 30-fold increase in translesion replication. Thus, similar to Escherichia coli DNA polymerase III, the processivity accessory proteins enable DNA polymerase delta to bypass blocking lesions. Under comparable conditions, DNA polymerase beta was unable to bypass the abasic site, unless its concentration was greatly increased. Analysis of translesion replication products revealed a marked difference in the specificity of bypass: whereas 90% of bypass events by DNA polymerase delta holoenzyme involved insertion of a dAMP residue opposite the abasic site, DNA polymerase beta tended to skip over the abasic site, producing mainly minus frameshifts (73%). The significance of these results for in vivo translesion replication is discussed.     

Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly

The bacteriophage T4 DNA polymerase holoenzyme, consisting of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62), is loaded onto DNA in an ATP-dependent, multistep reaction. The trimeric, ring-shaped gp45 is loaded onto DNA such that the DNA passes through the center of the ring. gp43 binds to this complex, thereby forming a topological link with the DNA and increasing its processivity. Using stopped-flow fluorescence-resonance energy transfer, we have investigated opening and closing of the gp45 ring during the holoenzyme assembly process. Two amino acids that lie on opposite sides of the gp45 subunit interface, W91 and V162C labeled with coumarin, were used as the fluorescence donor and acceptor, respectively. Free in solution, gp45 has two closed subunit interfaces with W91 to V162-coumarin distances of 19 A and one open subunit interface with a W91 to V162C-coumarin distance of 40 A. Making the assumption that the distance across the two closed subunit interfaces is unchanged during the holoenzyme assembly process, we have found that the distance across the open subunit interface is first increased to greater than 45 A and is then decreased to 30 A during a 10-step assembly mechanism. The gp45 ring is not completely closed in the holoenzyme complex, consistent with previous evidence suggesting that the C-terminus of gp43 is inserted into the gp45 subunit interface. Unexpectedly, ATP-hydrolysis events are coupled to only a fraction of the total distance change, with conformational changes linked to binding DNA and gp43 coupled to the majority of the total distance change. Using the nonhydrolyzable ATP analogue ATP-gamma-S results in formation of a nonproductive gp45 x gp44/62 complex; however, adding an excess of ATP to this nonproductive complex results in rapid ATP/ATP-gamma-S exchange to yield a productive gp45 x gp44/62 complex within seconds.     

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).     

Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.     

DNA replication and head assembly in bacteriophage T4.

Phage DNA was accumulated in cells of E. coli B, infected with the phage T4DtsLB3 (gene 42), without the synthesis of late proteins (in the presence of chloramphenicol). Then (stage II), chloramphenicol was removed and further replication of the phage DNA suppressed with hydroxyurea and by simultaneously raising the temperature to 40 degrees. The media M9 or M9 with 1% amino acid were used; the times of addition of chloramphenicol and the hydroxyurea concentration were also varied. It was also shown that in medium M9, at stage II, chiefly early proteins were synthesized. In the medium containing amino acids, at stage II the following was observed: 1) DNA synthesis was entirely suppressed and a degradation of DNA occurred; 2) both early and late proteins were synthesized, with a predominance of the latter; 3) an assembly of the elements of the phage tails and capsids occurred without the neck and flagellum, and a small number of phage particles were also found; 4) the capsids, isolated in a sucrose density gradient after lysis with chloroform, contained the proteins Palt, P20, P23, P24, several unidentified proteins, and did not contain Pwac, P23, and P22, 5) the yield of viable phage varied from 0.05 to 15% per cell. Thus, the entire morphogenesis of T4 phage can occur without accompanying replication of phage DNA.     

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and . Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.