DNA repair and the genetic effects of thymidylate stress in yeast

Folate antagonists, such as aminopterin, methotrexate and various sulfonamides, block de novo thymidylate biosynthesis in Saccharomyces cerevisiae. The resulting starvation for thymine nucleotides is lethal and recombinagenic in RAD wild-type strains. In this paper we report our studies of these effects in repair-deficient yeast. Antifolate treatment of various rad mutants revealed that repair defects influence the killing and recombination caused by thymidylate deprivation. Compared to a RAD wild-type strain, diploids homozygous for rad3, rad6 or rad18 were more resistant to cell killing. Thus, contrary to findings with conventional DNA-damaging agents, the lethal effects of thymidylate starvation appear to be ameliorated by certain DNA repair deficiencies. On the other hand, a rad50 strain was extremely sensitive to the antifolates. Within this series of diploids, increasing sensitivity to thymidylate starvation was accompanied by an increase in recombination frequencies. The degrees of lethality and recombination, induced by thymidylate depletion, were correlated with the severity of DNA-strand breakage in the RAD and rad50 strains. Experiments with diploids homozygous for rad52, rad54 or rad57 suggested that aborted recombination events, provoked by thymidylate deprivation, caused chromosome loss. Furthermore, the repair defects in these mutants indicated that double-strand breaks are among the lethal lesions induced by thymine nucleotide starvation. Finally, we discuss the possibility that the recombinagenicity of thymidylate stress may account for one type of acquired resistance to methotrexate in mammalian cells.     

Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

(Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity.     

Nuclear localization of de novo thymidylate biosynthesis pathway is required to prevent uracil accumulation in DNA

Uracil accumulates in DNA as a result of impaired folate-dependent de novo thymidylate biosynthesis, a pathway composed of the enzymes serine hydroxymethyltransferase (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase. In G1, this pathway is present in the cytoplasm and at S phase undergoes small ubiquitin-like modifier-dependent translocation to the nucleus. It is not known whether this pathway functions in the cytoplasm, nucleus, or both in vivo. SHMT1 generates 5,10-methylenetetrahydrofolate for de novo thymidylate biosynthesis, a limiting step in the pathway, but also tightly binds 5-methyltetrahydrofolate in the cytoplasm, a required cofactor for homocysteine remethylation. Overexpression of SHMT1 in cell cultures inhibits folate-dependent homocysteine remethylation and enhances thymidylate biosynthesis. In this study, the impact of increased Shmt1 expression on folate-mediated one-carbon metabolism was determined in mice that overexpress the Shmt1 cDNA (Shmt1tg+ mice). Compared with wild type mice, Shmt1tg+ mice exhibited elevated SHMT1 and TYMS protein levels in tissues and evidence for impaired homocysteine remethylation but surprisingly exhibited depressed levels of nuclear SHMT1 and TYMS, lower rates of nuclear de novo thymidylate biosynthesis, and a nearly 10-fold increase in uracil content in hepatic nuclear DNA when fed a folate- and choline-deficient diet. These results demonstrate that SHMT1 and TYMS localization to the nucleus is essential to prevent uracil accumulation in nuclear DNA and indicate that SHMT1-mediated nuclear de novo thymidylate synthesis is critical for maintaining DNA integrity.     

Serine hydroxymethyltransferase anchors de novo thymidylate synthesis pathway to nuclear lamina for DNA synthesis

The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.     

Determination of relative values of de novo biosynthesis and salvage pathway of thymidylate formation in rat decidual tissue

A method for the determination of relative values (%) of two pathways of thymidine-5'-phosphate (dTMP) formation, e.g. via de novo biosynthesis and through thymidine reutilization (salvage pathway), is proposed. It is shown that the relative values of dTMP formation through the salvage pathway in the mesometrial part of developing decidua in pregnant rats (9-11th day of ppregnancy) are 1.5-3.4 times higher as compared to those in the antimesometrial part. When dTMP biosynthesis is suppressed by aminopterine, up to 80% of total DNA thymind is synthesized at the expense of thymidine reutilization. The incorporation of 3H-thymidine into DNA was thereby increased approximately 8-fold irrespective of the decrease in the DNA synthesis rate (approximately 2.4 times). The dependence of the relative values of the thymidine reutilization pathway on the correlation of the thymidylate synthetase and thymidine kinase activities in the tissue is discussed. The ability of the cells to reutilize thymidine is interpreted in terms of their relative resistance to the effect of folic acid antagonists.     

Evidence for small ubiquitin-like modifier-dependent nuclear import of the thymidylate biosynthesis pathway

Perturbations in folate-mediated one-carbon metabolism increase rates of uracil misincorporation into DNA during replication, impair cellular methylation reactions, and increase risk for neural tube defects and cancer. One-carbon metabolism is compromised by folate deficiency and common genetic polymorphisms. In this study, the mechanism for the preferential partitioning of cytoplasmic serine hydroxymethyltransferase (cSHMT)-derived methylenetetrahydrofolate to de novo thymidylate biosynthesis was investigated. The cSHMT enzyme was shown to interact with UBC9 and was a substrate for UBC9-catalyzed small ubiquitin-like modifier (SUMO) modification in vitro. SUMOylated cSHMT was detected in extracts from S phase MCF-7 cells, and cSHMT was shown to localize to the nucleus and nuclear periphery during the S and G(2)/M phases of the cell cycle. A common single nucleotide polymorphism (L474F-cSHMT) impaired the UBC9-cSHMT interaction and inhibited cSHMT SUMOylation in vitro. The three folate-dependent enzymes that constitute the de novo thymidylate biosynthesis pathway, cSHMT, thymidylate synthase, and dihydrofolate reductase, all contain SUMO modification consensus sequences. Compartmentation of the folate-dependent de novo thymidylate biosynthesis pathway in the nucleus accounts for the preferential partitioning of cSHMT-derived folate-activated one-carbon units into thymidylate biosynthesis; the efficiency of nuclear folate metabolism is likely to be modified by the cSHMT L474F polymorphism.     

Effects of excess thymidylate on thymidylate low-requiring strains of Saccharomyces cerevisiae: high mutagenicity and absence of DNA strand breaks

dTMP exposure concentrations of 0.1 mM or higher are genotoxic in exponentially growing cells of thymidylate low-requiring mutants of Saccharomyces cerevisiae. Mutagenicity of excess dTMP is highest in an exposure concentration 10-fold of that needed for external supplementation of endogenously blocked thymidylate synthesis. Still higher dTMP concentrations are primarily cytotoxic. The canavanine forward-mutation system shows excess dTMP to be as potent a mutagen as irradiation by ultraviolet light. Mutagenicity of excess dTMP, however, differs from that of direct DNA-attacking mutagens in that it is highest in the absence of significant toxicity. Alkaline sucrose gradient centrifugation shows that excess dTMP does not induce significant numbers of DNA single- or double-strand breaks, while conditions of thymidylate deprivation lead to DNA-strand breaks and thymineless death.     

Exogenous dTMP utilization by a novel tup mutant of Saccharomyces cerevisiae.

The rate and extent of entry of dTMP were measured in strains of Saccharomyces cerevisiae carrying two new tup mutations (tup5 and tup7) and most of the other tup mutations which have been reported previously by others. The tup7 mutation allowed dramatically greater accumulation of dTMP than any of the other mutations tested. Specific labeling of DNA by [CH3-3H]dTMP, fate of the dTMP pool inside of the cells, and degradation of the dTMP in the culture medium were investigated in strains carrying the tup7 mutation. The extracellular dTMP was not appreciably degraded, and that accumulated intracellularly was readily phosphorylated to dTDP and dTTP. Under optimum labeling conditions, 60 to 80% of the total thymidylate residues in newly synthesized DNA were derived from the exogenously provided dTMP, even in the absence of a block in de novo dTMP biosynthesis. An apparent Km for entry of 2 mM dTMP was found. The tup7 mutation increased permeability to dTMP (and some other 5'-mononucleotides), but did not affect uptake of nucleosides and purine and pyrimidine bases. Uptake of dTMP could be almost completely inhibited by moderate concentrations of Pi. These findings and other observations suggest that entry of dTMP in strains carrying the tup7 mutation is mediated by a permease whose function in normal cells is the transport of Pi.     

Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886, Escherichia coli NC3, and Zymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing the thyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in the de novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression of thyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA in E. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA in E. coli would be expected to increase the cellular requirement for dTMP. Increased expression of thyA (E. coli, B. subtilis, or Z. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.     

A comparative study of the cytotoxic and genotoxic effects of cisplatin and its analogue, TNO-6, in yeast

The antitumour drug cisplatin and its analogue, TNO-6, were studied for their cytotoxic, mutagenic and recombinagenic effects in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. It was observed that the structural change in TNO-6 resulted in reduced cytotoxicity and recombinagenicity (mitotic gene conversion) but increased mutagenic activity compared to the effects of cisplatin at equimolar concentrations. These results indicated that the mechanism through which TNO-6 damages cellular DNA is different from that of cisplatin.     

Multiple mutant of Escherichia coli synthesizing virtually thymineless DNA during limited growth.

The dut gene of Escherichia coli encodes deoxyuridine triphosphatase, an enzyme that prevents the incorporation of dUTP into DNA and that is needed in the de novo biosynthesis of thymidylate. We produced a conditionally lethal dut(Ts) mutation and isolated a phenotypic revertant that had a mutation in an unknown gene tentatively designated dus (for dut suppressor). The dus mutation restored the ability of the dut mutant to grow at 42 degrees C without restoring its enzymatic activity or thymidylate independence. A strain was constructed bearing, in addition to these mutations, ones affecting the following genes and their corresponding products: ung, which produces uracil-DNA N-glycosylase, a repair enzyme that removes uracil from DNA; deoA, which produces thymidine (deoxyuridine) phosphorylase, which would degrade exogenous deoxyuridine; and thyA, which produces thymidylate synthase. When grown at 42 degrees C in minimal medium containing deoxyuridine, the multiple mutant displayed a 93 to 96% substitution of uracil for thymine in new DNA. Growth stopped after the cellular DNA had increased 1.6- to 1.9-fold and the cell mass had increased 1.7- to 2.7-fold, suggesting a general failure of macromolecular biosynthesis. DNA hybridization confirmed that the uracil-containing DNA was chromosomal and that new rounds of initiation must have occurred during its synthesis.     

Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell line as a tool for studying the thymidine salvage pathway and the incorporation of thymidine analogues into host cell DNA.

A thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-), auxotrophic for thymidine (dThd), proved extremely useful for studying the dependence of cell growth on the exogenous supply of dThd, the relation between cell growth and DNA synthesis, and the ability of a series of 25 5-substituted 2'-deoxyuridines (dUrd) to substitute for dThd in sustaining cell growth. FM3A/TS-cells did not proliferate unless dThd was supplied to the cell culture medium. The 5-halogenated dUrd derivatives 5-chloro-dUrd, 5-bromo-dUrd and 5-iodo-d Urd also sustained FM3A/TS- cell growth. The extents of incorporation of [methyl-3H]dThd and 5-iodo-[6-3H]dUrd into DNA were closely correlated with their stimulatory effects on FM3A/TS- cell growth. This suggests that the stimulatory effects of the dUrd analogues on the growth rate of FM3A/TS- cells may be considered as evidence for their incorporation into host cell DNA. Based on this premise it is postulated that, in addition to 5-chloro-dUrd, 5-bromo-dUrd, 5-iodo-dUrd and dThd itself, the following dThd analogues are also incorporated into FM3A/TS- cell DNA (in order of the extent to which they are incorporated): 5-hydroxy-dUrd greater than 5-propynyloxy-dUrd greater than 5-ethyl-dUrd greater than 5-ethynyl-dUrd approximately 5-vinyl-dUrd. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to dissociate the de novo and salvage pathways of dTMP biosynthesis and to distinguish those dUrd analogues that are incorporated into DNA from those that are not.     

The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits.

Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.     

Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus.

Thymidylate kinase (dTMP kinase; EC 2.7.4.9) catalyzes the phosphorylation of dTMP to form dTDP in both de novo and salvage pathways of dTTP synthesis. The nucleotide sequence of the tmk gene encoding this essential Escherichia coli enzyme is the last one among all the E. coli nucleoside and nucleotide kinase genes which has not yet been reported. By subcloning the 24.0-min region where the tmk gene has been previously mapped from the lambda phage 236 (E9G1) of the Kohara E. coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), we precisely located tmk between acpP and holB genes. Here we report the nucleotide sequence of tmk, including the end portion of an upstream open reading frame (ORF 340) of unknown function that may be cotranscribed with the pabC gene. The tmk gene was located clockwise of and just upstream of the holB gene. Our sequencing data allowed the filling in of the unsequenced gap between the acpP and holB genes within the 24-min region of the E. coli chromosome. Identification of this region as the E. coli tmk gene was confirmed by functional complementation of a yeast dTMP kinase temperature-sensitive mutant and by in vitro enzyme assay of the thymidylate kinase activity in cell extracts of E. coli by use of tmk-overproducing plasmids. The deduced amino acid sequence of the E. coli tmk gene showed significant similarity to the sequences of the thymidylate kinases of vertebrates, yeasts, and viruses as well as two uncharacterized proteins of bacteria belonging to Bacillus and Haemophilus species.     

Function and evolution of plasmid-borne genes for pyrimidine biosynthesis in Borrelia spp

The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.     

Raltitrexed’s effect on the development of neural tube defects in mice is associated with DNA damage,apoptosis, and proliferation

The causal metabolic pathway and the underlying mechanism between folate deficiency and neural tube defects (NTDs) remain obscure. Thymidylate (dTMP) is catalyzed by thymidylate synthase (TS) using the folate-derived one-carbon unit as the sole methyl donor. This study aims to examine the role of dTMP biosynthesis in the development of neural tube in mice by inhibition of TS via a specific inhibitor, raltitrexed (RTX). Pregnant mice were intraperitoneally injected with various doses of RTX on gestational day 7.5, and embryos were examined for the presence of NTDs on gestational day 11.5. TS activity and changes of dUMP and dTMP levels were measured following RTX treatment at the optimal dose. DNA damage was determined by detection of phosphorylated replication protein A2 (RPA2) and γ-H₂AX in embryos with NTDs induced by RTX. Besides, apoptosis and proliferation were also analyzed in RTX-treated embryos with NTDs. We found that NTDs were highly occurred by the treatment of RTX at the optimal dose of 11.5 mg/kg b/w. RTX treatment significantly inhibited TS activity. Meanwhile, dTMP was decreased associated with the accumulation of dUMP in RTX-treated embryos. Phosphorylated RPA2 and γ-H₂AX were significantly increased in RTX-treated embryos with NTDs compared to control. More apoptosis and decreased proliferation were also found in embryos with NTDs induced by RTX. These results indicate that impairment of dTMP biosynthesis caused by RTX led to the development of NTDs in mice. DNA damage and imbalance between apoptosis and proliferation may be potential mechanisms.     

Genotoxicity of excess thymidylate in thymidylate low-requiring Saccharomyces cerevisiae is associated with changes in phosphate metabolism

When dTMP in concentrations > 100 μM is offered to growing cells of thymidylate low-requiring yeast strains it is both mutagenic and toxic. At exposure concentrations > 1 mM dTMP interferes significantly with the low-affinity phosphate permease even in the presence of exogenous phosphate concentrations of 6 mM. Chemical analysis and ³¹P NMR spectroscopy reveal that excess dTMP distrubs metabolism in thymidylate low-requiring strains but not in the wild type. The most prominent changes in phosphorus-containing molecules are found in polyphosphates of which up to 20% are broken down within a 20-min time span with a concomitant increase in orthophosphate pools.     

Effects of pyrimidine salvage inhibitors on uracil incorporation of Toxoplasma gondii

Metabolic inhibitors which act in the process of pyrimidine salvage influenced on the uracil incorporation into nucleic acids of Toxoplasma. Inhibitors of dihydrofolate reductase, pyrimethamine and methotrexate, and inhibitors of thymidylate synthase, fluoro-uridine, fluoro-dUMP and fluoro-uracil, diminished isotopic uracil uptake in dose-dependent manners. Azauridine which suppresses de novo pyrimidine biosynthesis did not affect the salvage even in a relatively high dose. These results suggested that the activation of uracil salvage should be closely related with the function of TMP biosynthetic enzymes. The pattern of thymidine uptake had no differences between control HL-60 cells and Toxoplasma infected cells, which did not reflect the specific proliferation of Toxoplasma. It can be exploited to characterize the effects of various compounds related with the proliferation of Toxoplasma, especially its DNA synthesis.