Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase

Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene.     

Bacteriophage-coded thymidylate synthetase. Evidence that the T4 enzyme is a capsid protein

It has previously been shown that T4 bacteriophage-coded dihydrofolate reductase is a capsid protein, specifically an element of the tail plate. This paper presents evidence that thymidylate synthetase is also a structural protein. Antiserum prepared against purified T4 thymidylate synthetase neutralizes T4 infectivity. Evidence is presented that structural thymidylate synthetase is the target of the antiphage component of the serum.The td gene in T4 codes for thymidylate synthetase. We have crossed the td gene from phage T6 into T4 and eliminated other T6 genetic material from the hybrid phage by extensive backcrossing. The hybrid phage, T4td^T6, is inactivated at 60 °C significantly more rapidly than the parent phage, T4D. Thus, the td gene is a determinant of a physical property of the virion, providing direct confirmation that thymidylate synthetase is a capsid protein. At present the role of the virion-bound enzyme is unknown.     

Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens.

NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme.     

Induction of SOS responses in Escherichia coli by 5-fluorouracil

The inducibility of SOS responses by 5-fluorouracil (5-FU), which has been used as an antitumor drug, was studied in Escherichia coli cells which have different DNA repair capacities for UV lesions. Expression of the umuC gene was apparently induced by 5-FU in the wild-type and uvrA strains, but not in lexA and recA strains. The inducibility of the umuC gene by 5-FU, the metabolite of which inhibits thymidylate synthetase, was abolished in cultures containing deoxythymidine monophosphate which is converted from deoxyuridine monophosphate by thymidylate synthetase. These results suggest that 5-FU may exert its SOS inducibility by inhibiting thymidylate synthetase and then disturbing DNA metabolism but not by incorporating 5-FU residues into RNA. Further, 5-FU weakly induced mutations in E. coli.     

Thymidylate synthetase of human lymphocytes augmented in vitro by methotrexate

The addition of methotrexate to the assay system of thymidylate synthetase caused a reduction in the activity of the enzyme but addition of methotrexate to the culture of phytohemagglutinin stimulated normal human lymphocytes caused an increase in the activity of the enzyme which was abolished by the addition of actinomycin D or cycloheximide. These studies suggest that the antimetabolite augmented the enzyme activity by modulating the gene for the enzyme. This modulation of the gene could have been achieved by the thymineless state brought about by methotrexate or the antimetabolite could have affected gene reodont or brought about amplification of the gene. The results of the nucleoside incorporation were consistent with a thymidylate synthetase block; however, other explanations are offered.     

Human leukemic cells resistant to 5-fluoro-2'-deoxyuridine contain a thymidylate synthetase with lower affinity for nucleotides

A line of human lymphocytic leukemia cells (CCRF-CEM) has been obtained which is 140-fold resistant to the potent cell growth inhibitor 5-fluoro-2'-deoxyuridine (FdUrd). The cells were also 11-fold cross-resistant to 5-fluorouracil. In contrast to several previous studies involving FdUrd-resistant mouse cells, thymidylate synthetase levels were not substantially elevated in these FdUrd-resistant human leukemic cells. Thymidine kinase activity was also unchanged in the resistant cells, although the levels of 5-fluoro-2'-deoxyuridylate (FdUMP), the potent inhibitor of thymidylate synthetase, generated at equimolar doses of FdUrd were about 40% lower than in the sensitive cells. Studies of the kinetics of FdUMP binding to thymidylate synthetase isolated from the FdUrd-resistant cells disclosed a considerably higher dissociation constant (Kd = 1.0 X 10(-9) M) for the ternary covalent enzyme . FdUMP . 5,10-methylene tetrahydrofolate complex compared to the value obtained with enzyme from sensitive cells (Kd = 4.4 X 10(-11) M). The thymidylate synthetase from the FdUrd-resistant cells also showed 17-fold weaker binding of 2'-deoxyuridylate, even though the Km value for 2'-deoxyuridylate was 3-fold lower compared to the enzyme from FdUrd-sensitive cells. The turnover number of the altered enzyme was 1.8-fold higher than that for the normal enzyme but the rate constants for the release of FdUMP from the ternary complex, which is also an enzyme-catalyzed reaction, were identical for both enzymes. Electrophoresis of the radiolabeled ternary complexes on nondenaturing gels showed small but reproducible differences in migration rates. These results demonstrate that the mechanism of resistance to FdUrd in this cell line involves an alteration in the target enzyme, thymidylate synthetase, which causes it have a lower affinity for nucleotides.     

Inhibition of thymidylate synthetase of the human lymphocytes.

Previous studies had suggested that methotrexate (MTX) may have actions other than inhibition of dihydrofolic acid reductase. In this study MTX was added to the assay incubation mixture of the enzyme thymidylate synthetase. 5-fluorodeoxyuridine (FUdR) or folinic acid was added separately as controls. The three compounds inhibited thymidylate synthetase with MTX achieving the maximal inhibition. It is suggested that MTX could exert its antineoplastic effect through this mechanism especially if malignant cells have little or no activity of dihydrofolic acid reductase.     

A new immunoblotting assay for thymidylate synthetase and its application to the regulation of enzyme activity in regenerating rat liver

A highly sensitive and specific immunoblot assay has been developed to quantitate the content of rat liver thymidylate synthetase (EC 2.1.1.45). Applying the method, it is demonstrated that the increase of the activity of thymidylate synthetase in liver regeneration after partial hepatectomy is due to the de novo synthesis of the enzyme protein. Administration of cycloheximide, phenoxybenzamine, phorbol 12-myristate 13-acetate, nifedipine, dexamethasone or indomethacin to partially hepatectomized rats prevented the synthesis of thymidylate synthetase in regenerating liver. Thyroparathyroidectomy also inhibited the increase of the enzyme in liver regeneration. These observations are discussed in relation to the signal transduction concerning the alpha 1-receptor, which was shown to regulate liver regeneration in our previous papers.     

Assay of intracellular thymidylate synthetase activity and inhibition by 5-fluoro-2'-deoxyuridine in lymphocytes.

Thymidylate synthetase catalyses the formation of thymidine monophosphate from deoxyuridine monophosphate. Purified thymidylate synthetase can be assayed radiochemically using labelled deoxyuridine monophosphate as substrate, but cells are impervious to deoxyuridine monophosphate and so intracellular thymidylate synthetase activity cannot be assayed in this way. In this paper we describe the assay of intracellular thymidylate synthetase activity in intact cells using labelled 2'-deoxyuridine. The assay showed linear kinetics with respect to time, concentration of 2'-deoxyuridine, and cell concentration. 5-fluoro-2'-deoxyuridine inhibited intracellular thymidylate synthetase activity measured with this assay by 50% at 5 nM. Cell growth was inhibited by 50% at 6 nM 5-fluoro-2'-deoxyuridine. The assay was specific for thymidylate synthetase and enabled measurement of thymidylate synthetase activity in situ in intact cells.     

Kinetics of sulfhydryl group modification of thymidylate synthetase: a proposal for activation of catalytic cysteinly residues.

The pKa of ～8.0 for the catalytic cysteine residue of thymidylate synthetase was determined from the pH dependence of inactivation by the sulfhydryl reagents methyl methanethiolsulfonate and 5,5′ dithiobis (2-nitrobenzoic acid). At low pH (5.8–6.8) a rate of reaction significantly greater than can be accounted for by the concentration of thiolate anion was observed. The observed pKa and reactivity for thymidylate synthetase are comparable to those reported for papain (Little, G. L. and Brocklehurst, K. (1972) Biochem. J. $\text{128}$, 475–477) (1). On the basis of these observations we propose that the cysteines of thymidylate synthetase involved in covalent catalysis may be activated through interaction with a general base in the active site of the enzyme.     

The sex difference in the regulation of liver regeneration after partial hepatectomy in the rat

The increases in the activity of hepatic thymidylate synthetase and thymidine kinase, which catalyzes the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed 24 h after 70% partial hepatectomy in female rats administered either alpha- or beta-adrenoreceptor antagonists. The injection of beta-antagonist to male or ovariectomized female rats had no effect on the activities of these enzymes. Only alpha-adrenoceptor antagonist depressed these enzymatic activities of 24-h-regenerating liver in male and ovariectomized female rats. The decrease of the activities of thymidylate synthetase and thymidine kinase was accompanied by a concomitant reduction of DNA content in 24-h-regenerating liver. It is concluded that catecholamine regulates the female rat liver regeneration through both alpha- and beta-adrenergic pathways by the inductions of thymidylate synthase and thymidine kinase, while in adult male and ovariectomized female rats, only the alpha-mediated pathway is involved.     

Thymidylate synthetase inhibitors and fragile site expression in lymphocytes.

The ability of three thymidylate synthetase inhibitors, fluorodeoxyuridine, fluorodeoxycytidine, and trifluorothymidine, to induce the expression of eight different folate-sensitive fragile sites has been investigated in 22 patients and compared with the efficacy of simple folate deprivation for inducing fragile site expression. Fluorodeoxyuridine and fluorodeoxycytidine were equal in their ability to elicit fragile site expression but fluorodeoxycytidine proved less cytotoxic under comparable culture conditions. Both fluorodeoxyuridine and fluorodeoxycytidine were found to be more efficient than trifluorothymidine at comparable concentrations but less efficient than simple folate deprivation in eliciting fragile site expression in lymphocytes. Since the three inhibitors induced expression of eight different folate-sensitive fragile sites, it is likely that all folate-sensitive fragile sites have a common underlying mechanism of expression. The practical application of thymidylate synthetase inhibitors in the routine detection of heritable fragile sites is discussed.     

Differentiation associated changes in thymidylate synthetase and thymidine kinase activities in intestinal cells.

Proliferative and mature intestinal cells of the jejunum and colon of rat, colon of man, and the surface cells of neoplastic colon lesions of man were assayed for thymidylate synthetase and thymidine kinase activities. Cells from the proliferative region of rat jejunal mucosa were found to have higher enzyme activities than cells from the non-proliferative region. Thymidylate synthetase activity was observed to decrease as cells migrated from base to upper crypt, whereas thymidine kinase activity increased during crypt migration and then declined as cells migrated onto villi. Thymidine kinase activity also remained elevated longer than thymidylate synthetase during cell migration in colonic mucosa of rat and man. High thymidine kinase: thymidylate synthetase ratios similar to those observed in flat mucosa before cells become fully mature were found in cells removed from expanding neoplastic lesions of man.     

The effect of leucovorin on the therapeutic index of fluorouracil in cancer patients

Fluorouracil has been in clinical use as an anticancer drug for 30 years. Although this drug has a broad spectrum of anticancer activity, including significant activity against the common solid tumors of the gastrointestinal system, only a minority of patients treated with fluorouracil experience an objective response to therapy. Furthermore, in randomized clinical trials completed to date, it has not been possible to demonstrate that fluorouracil therapy significantly prolongs the life span of patients with advanced cancer. Recent laboratory studies have indicated that leucovorin can enhance the cytotoxicity of fluorouracil in vitro, evidently by enhancing inhibition of the key enzyme, thymidylate synthetase, by the fluorouracil metabolite, FdUMP (fluorodeoxyuridine monophosphate; a stable inactive FdUMP-reduced folate-thymidylate synthetase complex is formed). Pilot, uncontrolled studies of leucovorin-fluorouracil combinations have suggested that leucovorin may significantly increase both the clinical efficacy and the clinical toxicity of fluorouracil in cancer patients. These findings have led to the initiation of several randomized, controlled studies of leucovorin plus fluorouracil versus fluorouracil alone in the treatment of patients with advanced colorectal cancer. Three of these studies have recently completed patient accrual, and the preliminary results of each of the three studies indicate that leucovorin-fluorouracil combinations will have a better therapeutic index than fluorouracil used alone in this disease. Further follow-up of these studies will be needed to determine whether leucovorin-fluorouracil combination therapy will prolong the life span of patients with colorectal cancer.     

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.     

2'-deoxyribosyl analogues of UDP-N-acetylglucosamine in cells treated with methotrexate or 5-fluorodeoxyuridine

dUDP-GlcNAc, the 2'-deoxyribosyl analogue of UDP-GlcNAc, has been identified in human lymphoid cells treated with the dihydrofolate reductase inhibitor, methotrexate. It was shown previously that elevation of dUTP accompanies the gross expansion in intracellular deoxyuridylate pools that results from the methotrexate-induced block in thymidylate synthetase activity (1). dUDP-GlcNAc presumably is formed from dUTP acting in place of UTP in the normal pathway for formation of UDP-GlcNAc. Neither dUTP nor dUDP-GlcNAc has been detected in untreated cells. Inhibition of thymidylate synthetase by treatment of cells with 5-fluorodeoxyuridine (5-FdUrd) also causes the appearance of dUDP-GlcNAc, and, in addition, 5-FdUDP-GlcNAc, synthesized from 5-FdUTP. The metabolic effects, if any, of these analogues are not known. Synthesis of the analogues may help to limit accumulation of dUTP and 5-FdUTP under circumstances in which the deoxyuridine triphosphatase mechanism is insufficient.     

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.     

Thymidine-requiring mutants of Dictyostelium discoideum.

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.     

Quenching of thymidylate synthetase fluorescence by substrate analogs.

Quenching of fluorescence occurs when $\text{Lactobacillus casei}$ thymidylate synthetase is titrated with fluorodeoxyuridylate in the presence of 1-L-methylenetetrahydrofolate to form a ternary complex. Neither fluorodeoxyuridylate nor 1-L-methylenetetrahydrofolate added singly has any effect on enzyme fluorescence but d-L-methylenetetrahydrofolate alone causes quenching. Thus ternary complex formation and interaction with d-L-methylenetetrahydrofolate alter the environment of tryptophan residues in thymidylate synthetase in a similar manner.     

Relative affinity of 5,10-methylenetetrahydrofolylpolyglutamates for the Lactobacillus casei thymidylate synthetase-5-fluorodeoxyuridylate binary complex

The affinity of a series of methylenetetrahydrofolate polyglutamates for the binary complex of Lactobacillus casei thymidylate synthetase and fluorodeoxyuridylate has been investigated by kinetic and equilibrium techniques. The relative rates of binding for polyglutamates with one through seven glutamate residues were determined at 0 °C. Reactions were stopped by quenching into sodium dodecyl sulfate with subsequent determination of bound tritiated fluorodeoxyuridylate by gel filtration chromatography. Rates increased up to five residues beyond which a slight decrease occurred. Relative equilibrium binding affinities for all possible pairs of polyglutamates from the same series were determined by electrophoretic separation of tritiated complexes. In every case, the longer chain length member of the pair was bound more tightly. Complexes involving only a single subunit of thymidylate synthetase were compared with those in which both subunits were bound. Monomeric (1:1:1) complexes invariably showed a greater affinity for the longer chain length member of the pair than the corresponding dimeric (2:2:1) species. These results are interpreted in terms of possible binding site models for thymidylate synthetase.