The gene 1.2 protein of bacteriophage T7 interacts with the Escherichia coli dGTP triphosphohydrolase to form a GTP-binding protein

Escherichia coli encodes a dGTP triphosphohydrolase (dGTPase) that cleaves dGTP to deoxyguanosine and tripolyphosphate. dGTP is hydrolyzed with a Michaelis constant (Km) of 5 microM and a maximal velocity (Vmax) of 1.8 mumols/min/mg. The ribonucleotide GTP is a poor substrate with a much lower affinity. It is hydrolyzed with a Km of 150 microM and Vmax of 0.07 mumols/min/mg. Bacteriophage T7 encodes a specific inhibitor of dGTPase, the gene 1.2 protein, that forms a tight complex with the enzyme. The enzyme-inhibitor complex binds dGTP with a dissociation constant (KD) of 1.5 microM, but the bound dGTP is not hydrolyzed. It remains stably bound to the complex with a half-life of approximately 5 min. In contrast, dGTP is unable to bind to gene 1.2 protein alone, and dGTP bound to dGTPase alone is quickly hydrolyzed and released. Surprisingly, the dGTPase-gene 1.2 protein complex has a higher affinity for GTP than for dGTP. GTP is stably bound to the dGTPase-gene 1.2 protein complex with a half-life greater than 30 min and KD of 0.8 microM; GTP is not stably bound to either dGTPase or gene 1.2 protein alone. Both GTP and dGTP bind to and stabilize the dGTPase-gene 1.2 protein complex, inhibiting its dissociation. Although the presence of dGTP induces conformation changes in dGTPase so that it is unable to associate with the gene 1.2 protein, saturating concentrations of GTP have no such effect. The enzyme efficiently associates with its inhibitor in the presence of GTP. These results indicate that E. coli dGTPase and gene 1.2 protein interact to form a high affinity GTP-binding site. dGTP is most effective in preventing the association of the enzyme with the inhibitor whereas GTP is most effective in preventing the dissociation of the enzyme-inhibitor complex.     

Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools

The gene 1.2 protein of bacteriophage T7, a protein required for phage T7 growth on Escherichia coli optA1 strains, has been purified to apparent homogeneity and shown to restore DNA packaging activity of extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants (Myers, J. A., Beauchamp, B. B., White, J. H., and Richardson, C. C. (1987) J. Biol. Chem. 262, 5280-5287). After infection of E. coli optA1 by T7 gene 1.2 mutant phage, under conditions where phage DNA synthesis is blocked, the intracellular pools of dATP, dTTP, and dCTP increase 10-40-fold, similar to the increase observed in an infection with wild-type T7. However, the pool of dGTP remains unchanged in the mutant-infected cells as opposed to a 200-fold increase in the wild-type phage-infected cells. Uninfected E. coli optA+ strains contain severalfold higher levels of dGTP compared to E. coli optA1 cells. In agreement with this observation, dGTP can fully substitute for purified gene 1.2 protein in restoring DNA packaging activity to extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants. dGMP or polymers containing deoxyguanosine can also restore packaging activity while dGDP is considerably less effective. dATP, dTTP, dCTP, and ribonucleotides have no significant effect. The addition of dGTP or dGMP to packaging extracts restores DNA synthesis. Gene 1.2 protein elevates the level of dGTP in these packaging extracts and restores DNA synthesis, thus suggesting that depletion of a guanine deoxynucleotide pool in E. coli optA1 cells infected with T7 gene 1.2 mutants may account for the observed defects.     

Location and molecular cloning of the structural gene for the deoxyguanosine triphosphate triphosphohydrolase of Escherichia coli

The structural gene for deoxyguanosine triphosphate triphosphohydrolase (dGTPase) (EC 3.1.5.1) and its regulator, optA, have been located on a lambda phage carrying a 17.5kb Escherichia coli DNA insert. The DNA fragment has been excised and ligated into pBR325 and also transferred to another lambda vector. From the results of transduction and transformation experiments, we find that the structural gene for dGTPase is very closely linked to optA and dapD, which locates it at approximately 3.6 minutes on the genetic map of E. coli K12. We propose the mnemonic dgt as the designation for the structural gene for this enzyme.     

Primary structure of the deoxyguanosine triphosphate triphosphohydrolase-encoding gene (dgt) of Escherichia coli

The complete nucleotide sequence has been determined for a 2027-bp region that encompasses the structural gene (dgt) encoding deoxyguanosine triphosphate triphosphohydrolase (dGTPase) from Escherichia coli. The gene resides between the htrA and dapD loci at 3.75-3.8' on the bacterial chromosome. Using homologous recombination in a recD recipient, a dgt- bacterial strain was constructed that was deficient in producing functional dGTPase. Comparison of dGTP pools in this and other strains revealed that dGTPase synthesized in vivo does to some degree modulate the level of dGTP in the bacterial cell, yet the magnitude of this modulation may be insufficient to explain the physiological function of dGTPase.     

The purification and properties of deoxyguanosine triphosphate triphosphohydrolase from Escherichia coli

Deoxyguanosine triphosphate (dGTP) triphosphohydrolase (EC 3.1.5.1) has been purified approximately 16,000-fold to apparent homogeneity from extracts of Escherichia coli. The enzyme has a native molecular weight of 230,000 and a sedimentation coefficient of 9.3 S. Its subunit molecular weight derived from electrophoresis in denaturing polyacrylamide gels is 58,900, and it has a unique N-terminal sequence for the first 25 amino acids, which indicate that the native enzyme is composed of 4 homologous subunits. It is insensitive to sulfhydryl reagents and EDTA and can be heated to 60 degrees C for 60 min without loss of activity. The enzyme requires Mg2+ for activity, is highly specific for dGTP among the canonical deoxynucleoside triphosphates, and has a unique activity among nucleoside triphosphatases in that the products of the reaction are deoxyguanosine and inorganic tripolyphosphate. Preliminary evidence suggest that this enzyme is responsible for the optA mutant phenotype first described by Saito and Richardson (Saito, H., and Richardson, C.C. (1981) J. Virol. 37, 343-351).     

Structure of Escherichia coli dGTP Triphosphohydrolase: A HEXAMERIC ENZYME WITH DNA EFFECTOR MOLECULES*

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present study, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNA with high affinity (K_d ∼ 50 nm). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent K_m for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.     

Purification and characterization of the gene 1.2 protein of bacteriophage T7

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.     

Genetic analysis of gene 1.2 of bacteriophage T7: isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants.

The product of gene 1.2 of bacteriophage T7 is not required for the growth of T7 in wild-type Escherichia coli since deletion mutants lacking the entire gene 1.2 grow normally (Studier et al., J. Mol. Biol. 135:917-937, 1979). By using a T7 strain lacking gene 1.2, we have isolated a mutant of E. coli that was unable to support the growth of both point and deletion mutants defective in gene 1.2. The mutation, optA1, was located at approximately 3.6 min on the E. coli linkage map in the interval between dapD and tonA; optA1 was 92% cotransducible with dapD. By using the optA1 mutant, we have isolated six gene 1.2 point mutants of T7, all of which mapped between positions 15 and 16 on the T7 genetic map. These mutations have also been characterized by DNA sequence analysis, E. coli optA1 cells infected with T7 gene 1.2 mutants were defective in T7 DNA replication; early RNA and protein synthesis proceeded normally. The defect in T7 DNA replication is manifested by a premature cessation of DNA synthesis and degradation of the newly synthesized DNA. The defect was not observed in E. coli opt+ cells infected with T7 gene 1.2 mutants or in E. coli optA1 cells infected with wild-type T7 phage.     

The dgt gene of Escherichia coli facilitates thymine utilization in thymine-requiring strains

The Escherichia coli dGTP triphosphohydrolase (dGTPase) encoded by the dgt gene catalyses the hydrolysis of dGTP to deoxyguanosine and triphosphate. The recent discovery of a mutator effect associated with deletion of dgt indicated participation of the triphosphohydrolase in preventing mutagenesis. Here, we have investigated the possible involvement of dgt in facilitating thymine utilization through its ability to provide intracellular deoxyguanosine, which is readily converted by the DeoD phosphorylase to deoxyribose-1-phosphate, the critical intermediate that enables uptake and utilization of thymine. Indeed, we observed that the minimal amount of thymine required for growth of thymine-requiring (thyA) strains decreased with increased expression level of the dgt gene. As expected, this dgt-mediated effect was dependent on the DeoD purine nucleoside phosphorylase. We also observed that thyA strains experience growth difficulties upon nutritional shift-up and that the dgt gene facilitates adaptation to the new growth conditions. Blockage of the alternative yjjG (dUMP phosphatase) pathway for deoxyribose-1-phosphate generation greatly exacerbated the severity of thymine starvation in enriched media, and under these conditions the dgt pathway becomes crucial in protecting the cells against thymineless death. Overall, our results suggest that the dgt-dependent pathway for deoxyribose-1-phosphate generation may operate under various cell conditions to provide deoxyribosyl donors.     

Kinetics of incorporation of O6-methyldeoxyguanosine monophosphate during in vitro DNA synthesis

O6-Methyldeoxyguanosine triphosphate (m6dGTP), known to be produced in vivo by methylation of deoxyguanosine triphosphate with simple methylating mutagens, is utilized by prokaryotic DNA polymerases during in vitro replication of synthetic and natural DNA template-primers. A study of the kinetic behavior of m6dGTP during DNA replication in vitro and of its effect on DNA replication indicates that m6dGTP acts as an analogue of dATP with Kappm of about 6 microM for Escherichia coli DNA polymerase I (Klenow fragment) compared to the Kappm of about 0.8 microM for dATP. m6dGTP is not incorporated in the complete absence of dATP (a competitive inhibitor). m6dGTP also inhibits in vitro DNA synthesis. Different DNA polymerases behave differently in utilization and turnover of m6dGTP. T4 DNA polymerase shows stronger discrimination against m6dGMP incorporation than either T5 DNA polymerase or E. coli DNA polymerase I. The possibility that m6dGTP is unlikely to contribute significantly to in vivo mutation is discussed.     

Mutations within the putative active site of heterodimeric deoxyguanosine kinase block the allosteric activation of the deoxyadenosine kinase subunit

Replacement of the Asp-84 residue of the deoxyguanosine kinase subunit of the tandem deoxyadenosine kinase/ deoxyguanosine kinase (dAK/dGK) from Lactobacillus acidophilus R-26 by Ala, Asn, or Glu produced increased Km values for deoxyguanosine on dGK. However, it did not seem to affect the binding of Mg-ATP. The Asp-84 dGK replacements had no apparent effect on the binding of deoxyadenosine by dAK. However, the mutant dGKs were no longer inhibited by dGTP, normally a potent distal endproduct inhibitor of dGK. Moreover, the allosteric activation of dAK activity by dGTP or dGuo was lost in the modified heterodimeric dAK/dGK enzyme. Therefore, it seems very likely that Asp-84 participates in dGuo binding at the active site of the dGK subunit of dAK/dGK from Lactobacillus acidophilus R-26.     

The mechanism of inhibition and "reversal" of mitogen-induced lymphocyte activation in a model of purine-nucleoside phosphorylase deficiency

Purine-nucleoside phosphorylase (PNP) is a purine degradative enzyme that catalyzes the phosphorolysis of (deoxy) inosine or (deoxy) guanosine to their respective bases and (deoxy) ribose 1-phosphate. A severe T-cell immune deficiency syndrome with hypouricemia is associated with impaired PNP function. To study the biochemical basis for this syndrome we created an in vitro model of PNP deficiency in mitogen (phytohemagglutinin)-stimulated normal human peripheral blood lymphocytes using guanosine to competitively inhibit deoxyguanosine phosphorolysis. Guanosine-induced guanine toxicity was reversed by adenine. Under these conditions, deoxyguanosine (5-45 microM) diminished mitogen stimulation to 30% of control while increasing the deoxyguanosine triphosphate pool (dGTP) by over 20-fold. Deoxycytidine reversed deoxyguanosine toxicity with a diminution of dGTP accumulation, but no significant change in the deoxycytidine triphosphate pool. Thymidine reversed the deoxyguanosine toxicity, repleted the thymidine triphosphate (dTTP) pool, and caused an even further increase in the accumulation of dGTP. These data support a model of lymphotoxicity in PNP deficiency based on dGTP accumulation with inhibition of ribonucleotide reductase and depletion of the thymidine triphosphate pool. Thymidine triphosphate depletion is reversed by either deoxycytidine or thymidine; however, the former diminishes dGTP accumulation (probably by competition for phosphorylation) and the latter potentiates dGTP accumulation (probably through feedback augmentation of guanosine diphosphate (GDP) reduction by ribonucleotide reductase secondary to an increased dTTP pool).     

dGTP triphosphohydrolase, a unique enzyme confined to members of the family Enterobacteriaceae.

The enzyme dGTP triphosphohydrolase (dGTPase; EC 3.1.5.1) was assayed in partially purified extracts of several genera of bacteria, and it was found to be strictly confined to members of the family Enterobacteriaceae. Whereas 11 of 12 enteric bacteria had comparable activity for this enzyme, 8 of 8 nonenteric bacteria, including species in the very closely related genera Vibrio and Aeromonas, did not assay positively for this enzyme. When challenged with Escherichia coli anti-dGTPase antiserum, the active enzymes fell into three groups, retaining 0, approximately 50, or 100% of their original activity. A computer search has revealed an amino acid sequence in the E. coli enzyme which matches well with the single-stranded-DNA binding motif of Prasad and Chiu (J. Mol. Biol. 193:579-584, 1987) and may account for the enzyme's observed interaction with DNA. As far as we are aware, this is the only enzymatic activity so far reported to be present solely in the enteric bacteria.     

Mechanisms of deoxyguanosine lymphotoxicity. Human thymocytes, but not peripheral blood lymphocytes accumulate deoxy-GTP in conditions simulating purine nucleoside phosphorylase deficiency

This study was designed to simulate purine nucleoside phosphorylase (PNP) deficiency by preincubating with guanosine (Guo) to minimize PNP activity while investigating the metabolism of [14C] deoxyguanosine (dGuo) at physiologic concentrations (10 microM) by unstimulated thymocytes, tonsil-derived T and B lymphocytes, and peripheral blood cells over short time periods. GTP was the principal metabolite formed from dGuo by all cell types with functional PNP and hypoxanthine-guanine phosphoribosyltransferase, confirming formation via degradation to guanine with subsequent salvage by hypoxanthine-guanine phosphoribosyltransferase. Thymocytes also formed a small amount of deoxyguanosine triphosphate (dGTP), presumably through direct phosphorylation by deoxycytidine kinase. Incorporation of dGuo into GTP was effectively inhibited in all instances under PNP deficiency conditions and dGTP levels increased up to 10-fold in thymocytes, but tonsil-derived B or T lymphocytes and unfractionated PBL still accumulated no detectable dGTP. E and platelets formed low amounts of dGTP under these conditions. Preincubation with adenine (50 microM) to reverse any Guo-induced toxicity reduced the incorporation of dGuo into GTP without inhibitor in all cell types with intact adenine phosphoribosyltransferase, but had no effect on dGTP accumulation in thymocytes, with or without inhibitor, thus excluding any indirect formation of dGTP via the de novo route. The rapid metabolism of dGuo to GTP, in the absence of PNP inhibition and subsequent effects of the altered GTP concentrations on cellular metabolism, may account for the differing responses reported by investigators with the use of low dGuo concentrations (enhancing), compared with high (inhibitory), concentrations in mitogen-stimulated lymphocyte studies. The exclusive ability of thymocytes to accumulate significant amounts of dGTP, and inability of B cells to do so, provides a logical explanation for the selective T cell immunodeficiency in PNP deficiency.     

Deoxynucleoside Kinases of Bacillus megaterium KM

Dialyzed extracts of Bacillus megaterium KM contain thymidine, deoxyadenosine, and deoxyguanosine kinase activities. Thymidine kinase activity is best with deoxyadenosine triphosphate or deoxyguanosine triphosphate (dGTP) as the phosphoryl donor, whereas the best deoxyadenosine kinase activity is obtained with dGTP or adenosine triphosphate. Deoxyguanosine kinase activity functions optimally with deoxycytidine triphosphate as the donor. Although the thymidine kinase activity of crude extracts does not have a demonstrable divalent cation requirement, the addition of Mg(2+) or Mn(2+) is necessary for the formation of thymidine di- and triphosphates. The synthesis of thymidine kinase appears to be partially derepressed by thymine starvation. Incubation of extracts with deoxyadenosine and dGTP results in the substantial accumulation of deoxyadenosine di- and triphosphates. Extracts deaminate deoxycytidine to deoxyuridine, presumably as a consequence of the action of deoxycytidine deaminase, and then convert deoxyuridine to deoxyuridylic acid. B. megaterium extracts do not contain any detectable deoxycytidine kinase activity.     

Characterization of a nucleotide kinase encoded by bacteriophage t7

Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ～12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) m obtained with dTTP for dTMP kinase is ～3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) m), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.     

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.     

High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP

Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain–containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80–2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1–2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.     

An effect of enzyme and ligand concentration on the state of aggregation of aspartate transcarbamylase of E. coli: I. The binding of CTP and ATP to the enzyme.

Detailed binding studies of the inhibitor, cytidine triphosphate (CTP), to native Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) reveal significant changes in subunit interaction when enzyme concentration is altered. In contrast, similar binding studies of the activator, adenosine triphosphate (ATP), do not reveal such changes, but do indicate more complex subunit interactions than previously reported. Equilibrium dialysis studies of 4 degrees C are consistent with six binding sites for CTP and ATP per enzyme molecule of molecular weight 310 000, at all enzyme concentrations. CTP binding studies reveal a progressive change from apparent positive to negative cooperativity as the enzyme concentration is decreased. ATP binding studies reveal complex subunit interactions involving a mixture of apparent negative and positive cooperativity. Sucrose gradient studies indicate the presence of at least three enzymatically active polymeric forms of the enzyme. The preliminary sedimentation studies indicate possible ligand and enzyme concentration perturbations of a preexisting association equilibrium in the aspartate transcarbamylase system. The binding data are therefore interpreted in terms of an association model.     

Effect of DNA sequence and structure on nuclease activity of the DexA protein of bacteriophage T4.

The bacteriophage T4 dexA gene product is required during infection of Escherichia coli strains carrying a mutation in the optA gene. We purified the DexA protein from cells which overproduced the protein. The protein was assayed for nuclease activity on synthetic di- and oligonucleotide substrates of known sequence and secondary structure. Sequence and structure significantly affected nuclease activity. The properties of the enzyme may explain the requirement for the DexA protein during infection of optA mutant hosts.