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.     

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.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

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.     

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.     

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.     

Multiple ATP binding is required to stabilize the "activated" (clamp open) clamp loader of the T4 DNA replication complex

Most DNA replication systems include a sliding clamp that encircles the genomic DNA and links the polymerase to the template to control polymerase processivity. A loading complex is required to open the clamp and place it onto the DNA. In phage T4 this complex consists of a trimeric clamp of gp45 subunits and a pentameric loader assembly of four gp44 and one gp62 subunit(s), with clamp loading driven by ATP binding. We measure this binding as a function of input ligand concentration and show that four ATPs bind to the gp44/62 complex with equal affinity. In contrast, the ATPase rate profile of the clamp-clamp loader complex exhibits a marked peak at an input ATP concentration close to the overall Kd (approximately 30 microm), with further increases in bound ATP decreasing the ATPase rate to a much lower level. Thus the progressive binding of the four ATPs triggers a conformational change in the complex that markedly inhibits ATPase activity. This inhibition is related to ring opening by using a clamp that is covalently cross-linked across its subunit interfaces and thus rendered incapable of opening. Binding of this clamp abolishes substrate inhibition of the ATPase but leaves ATP binding unchanged. We show that four ATP ligands must bind to the T4 clamp loader before the loader can be fully "activated" and the clamp opened, and that ATP hydrolysis is required only for release of the loader complex after clamp loading onto the replication fork has been completed.     

Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template.

T4 DNA polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA-binding protein catalyze ATP-dependent DNA synthesis. Using DNA primers with cross-linkable residues at specific positions, we obtained structural data that reveal how these proteins assemble on the primer-template. With the nonhydrolyzable ATP analog ATP gamma S, assembly of the 44/62 and 45 proteins on the primer requires 32 protein but not polymerase. ATP hydrolysis changes the position and intensity of cross-linking to each of the accessory proteins and allows cross-linking of polymerase. Our data indicate that the initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of the accessory proteins to yield a complex capable of processive DNA synthesis.     

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.     

PilB and PilT are ATPases acting antagonistically in type IV pilus function in Myxococcus xanthus

Type IV pili (T4P) are dynamic surface structures that undergo cycles of extension and retraction. T4P dynamics center on the PilB and PilT proteins, which are members of the secretion ATPase superfamily of proteins. Here, we show that PilB and PilT of the T4P system in Myxococcus xanthus have ATPase activity in vitro. Using a structure-guided approach, we systematically mutagenized PilB and PilT to resolve whether both ATP binding and hydrolysis are important for PilB and PilT function in vivo. PilB as well as PilT ATPase activity was abolished in vitro by replacement of conserved residues in the Walker A and Walker B boxes that are involved in ATP binding and hydrolysis, respectively. PilB proteins containing mutant Walker A or Walker B boxes were nonfunctional in vivo and unable to support T4P extension. PilT proteins containing mutant Walker A or Walker B boxes were also nonfunctional in vivo and unable to support T4P retraction. These data provide genetic evidence that both ATP binding and hydrolysis by PilB are essential for T4P extension and that both ATP binding and hydrolysis by PilT are essential for T4P retraction. Thus, PilB and PilT are ATPases that act at distinct steps in the T4P extension/retraction cycle in vivo.     

Modulation of cytoplasmic dynein ATPase activity by the accessory subunits

The microtubule-based motor molecule cytoplasmic dynein has been proposed to be regulated by a variety of mechanisms, including phosphorylation and specific interaction with the organelle-associated complex, dynactin. In this study, we examined whether the intermediate chain subunits of cytoplasmic dynein are involved in modulation of ATP hydrolysis, and thereby affect motility. Treatment of testis cytoplasmic dynein under hypertonic salt conditions resulted in separation of the intermediate chains from the remainder of the dynein molecule, and led to a 4-fold enhancement of ATP hydrolysis. This result suggests that the accessory subunits act as negative regulators of dynein heavy chain activity. Comparison of ATPase activities of dyneins with differing intermediate chain isoforms showed significant differences in basal ATP hydrolysis rates, with testis dynein 7-fold more active than dynein from brain. Removal of the intermediate chain subunits led to an equalization of ATPase activity between brain and testis dyneins, suggesting that the accessory subunits are responsible for the observed differences in tissue activity. Finally, our preparative procedures have allowed for the identification and purification of a 1:1 complex of dynein with dynactin. As this interaction is presumed to be mediated by the dynein intermediate chain subunits, we now have defined experimental conditions for further exploration of dynein enzymatic and motility regulation.     

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-dependent adenosinetriphosphatase C1 from mouse FM3A cells has DNA helicase activity.

In our previous study, we identified four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, in mouse FM3A cells (Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533). The DNA-dependent ATPase C1 has been purified and characterized in detail. A divalent cation and a polynucleotide cofactor were required for the ATPase activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Almost no ATPase activity was observed with S1 nuclease-treated native DNA. ATPase C1 hydrolyzed ATP only among the ribo- and deoxyribonucleoside triphosphates tested, and this fact distinguished ATPase C1 from ATPases B, C2, and C3, because the latter enzymes are capable of hydrolyzing both ATP and dATP. The purified DNA-dependent ATPase C1 fraction was shown to have a DNA helicase activity that was dependent on hydrolysis of ATP. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.2 S on glycerol gradient centrifugation. Both activities showed similar preferences for nucleoside 5'-triphosphates and similar requirements for divalent cations. The DNA helicase activity was inhibited by the addition of single-stranded DNAs that served as cofactor for the ATPase activity. The efficiency of a single-stranded DNA to inhibit DNA helicase activity correlated well with the capacity of the DNA to serve as cofactor for DNA-dependent ATPase activity. The helicase was shown to migrate along the DNA strand in the 5' to 3' direction, which is the same direction of migration of the mouse DNA helicase B (Seki, M., Enomoto, T., Yanagisawa, J., Hanaoka, F., and Ui, M. (1988) Biochemistry 27, 1766-1771).     

ATPase activity of the terminase subunit pUL56 of human cytomegalovirus.

Herpesviral DNA packaging is a complex process resulting in unit-length genomes packed into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of double-stranded DNA bacteriophages, the translocation of DNA was shown to be an energy-dependent process associated with an ATPase activity of the large terminase subunit. In the case of human cytomegalovirus it was not known which protein has the ability to hydrolyze ATP. In this study we expressed human cytomegalovirus terminase subunits, pUL89 and the carboxyl-terminal half of pUL56, as GST fusion proteins and purified these by affinity chromatography. ATPase assays demonstrated that the enzymatic activity is exclusively associated with pUL56. The characterization of the ATP hydrolysis showed that the enzymatic reaction is a fast process, whereas the spontaneous ATP decay followed slow kinetics. Interestingly, although pUL89 did not show any ATPase activity, it was capable of enhancing the UL56-associated ATP hydrolysis. Furthermore, a specific association of in vitro translated pUL89 with the carboxyl-terminal half of GST-UL56C was detected. This interaction was confirmed by co-immunoprecipitations of infected cells. Our results clearly demonstrated that (i) both terminase subunits interact with each other and (ii) the subunit pUL56 has an ATPase activity.     

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.     

Characterization of the five replication factor C genes of Saccharomyces cerevisiae.

Replication factor C (RFC) is a five-subunit DNA polymerase accessory protein that functions as a structure-specific, DNA-dependent ATPase. The ATPase function of RFC is activated by proliferating cell nuclear antigen. RFC was originally purified from human cells on the basis of its requirement for simian virus 40 DNA replication in vitro. A functionally homologous protein complex from Saccharomyces cerevisiae, called ScRFC, has been identified. Here we report the cloning, by either peptide sequencing or by sequence similarity to the human cDNAs, of the S. cerevisiae genes RFC1, RFC2, RFC3, RFC4, and RFC5. The amino acid sequences are highly similar to the sequences of the homologous human RFC 140-, 37-, 36-, 40-, and 38-kDa subunits, respectively, and also show amino acid sequence similarity to functionally homologous proteins from Escherichia coli and the phage T4 replication apparatus. All five subunits show conserved regions characteristic of ATP/GTP-binding proteins and also have a significant degree of similarity among each other. We have identified eight segments of conserved amino acid sequences that define a family of related proteins. Despite their high degree of sequence similarity, all five RFC genes are essential for cell proliferation in S. cerevisiae. RFC1 is identical to CDC44, a gene identified as a cell division cycle gene encoding a protein involved in DNA metabolism. CDC44/RFC1 is known to interact genetically with the gene encoding proliferating cell nuclear antigen, confirming previous biochemical evidence of their functional interaction in DNA replication.     

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.     

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.     

Structural biochemistry of ATP-driven dimerization and DNA-stimulated activation of SMC ATPases

Structural maintenance of chromosome (SMC) proteins play a central role in higher-order chromosome structure in all kingdoms of life. SMC proteins consist of a long coiled-coil domain that joins an ATP binding cassette (ABC) ATPase domain on one side and a dimerization domain on the other side. SMC proteins require ATP binding or hydrolysis to promote cohesion and condensation, which is suggested to proceed via formation of SMC rings or assemblies. To learn more about the role of ATP in the architecture of SMC proteins, we report crystal structures of nucleotide-free and ATP bound P. furiosus SMC ATPase domains. ATP dimerizes two SMC ATPase domains by binding to opposing Walker A and signature motifs, indicating that ATP binding can directly assemble SMC proteins. DNA stimulates ATP hydrolysis in the engaged SMC ABC domains, suggesting that ATP hydrolysis can be allosterically regulated. Structural and mutagenesis data identify an SMC protein conserved-arginine finger that is required for DNA stimulation of the ATPase activity and directly connects a putative DNA interaction site to ATP. Our results suggest that stimulation of the SMC ATPase activity may be a specific feature to regulate the ATP-driven assembly and disassembly of SMC proteins.