Quantitation of Some DNA Precursor Data

THE work of Kornberg on DNA repair and synthesis^1,2 implicates deoxyribonucleoside 5′-triphosphate as a direct precursor of DNA synthesis. This relationship was questioned by the possibility of alternative replication schemes^3,4. Werner⁵ studied the flux of thymine and thymidine into Escherichia coli DNA to determine the in vivo precursors of replicating DNA. Werner studied the incorporation of ³H labelled thymine into DNA and intracellular nucleotide pools under steady state conditions, in which thymine is converted into thymidine, thymidine monophosphate (TMP), thymidine diphosphate (TDP) and thymidine triphosphate (TTP). Werner measured separately the activities of labelled TMP, TDP, TTP and DNA at various times after E. coli cells had been exposed to a ³H-thymine synthetic medium. From a qualitative consideration of his results, Werner concluded that both TDP and TTP—but not TMP—were possible direct precursors of DNA replication.     

DNA synthesis in permeabilized WI38 and MRC5 cells

DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident.     

NADH-dependent O-deethylation of p-nitrophenetole with rabbit liver microsomes.

A previous paper in this series (C. K. Mathews, (1972) J. Biol. Chem.247, 7430) showed that deoxynucleoside triphosphate pools expand manyfold when DNA synthesis is blocked genetically in infection by bacteriophage T4. This paper describes a more detailed analysis of this phenomenon. The key approach involves labeling with thymine or thymidine under conditions of infection where both phage and host bear mutations that inactivate thymidylate synthetase. Principal findings include the following: (1) Nucleotides in the expanded pools are derived in roughly equal measure from breakdown of host cell DNA and from nucleotide synthesis de novo after infection. (2) Thymidine diphosphate pool expansion is comparable, in rate and extent, to thymidine triphosphate pool expansion, but thymidine monophosphate pools accumulate much less. (3) The rate of expansion of the total thymine nucleotide pool following temperature upshift in infection by a temperature-sensitive gene 45 mutant is approximately equal to the rate of thymine incorporation into DNA immediately preceding the upshift. (4) Similarly, when DNA synthesis is restored by a downshift, the total thymine nucleotide pool drains at a rate commensurate with that of thymine incorporation into DNA. (5) Under these latter conditions the dTTP pool begins to drain earlier than the dTDP pool, suggesting that dTTP is the more proximal DNA precursor in this system.     

New Deoxyribonucleic Acid Polymerase Induced by Bacillus subtilis Bacteriophage PBS2

The deoxyribonucleic acid (DNA) of Bacillus subtilis phage PBS2 has been confirmed to contain uracil instead of thymine. PBS2 phage infection of wild-type cells or DNA polymerase-deficient cells results in an increase in the specific activity of DNA polymerase. This induction of DNA polymerase activity is prevented by actinomycin D and chloramphenicol. In contrast to the major B. subtilis DNA polymerase, which prefers deoxythymidine triphosphate (dTTP) to deoxyuridine triphosphate (dUTP), the DNA polymerase in crude extracts of PBS2-infected cells is equally active whether dTTP or dUTP is employed. This phage-induced polymerase may be responsible for the synthesis of uracil-containing DNA during PBS2 phage infection.     

An enzymatic method for distinguishing deoxyuridine and deoxythymidine nucleotide pools and its application for determining ribonucleotide reductase activity.

A method is described for distinguishing deoxyuridine and deoxythymidine di- and triphosphate pools. The method utilizes a DNA polymerase assay for triphosphate determination and a coupled assay in which the disphosphate is converted to its corresponding triphosphate by nucleoside-diphosphate kinase and the triphosphate is measured by the DNA polymerase assay. By including deoxyruidine-triphosphate nucleotidohydrolase in the reaction mixture, dUTP is removed as a substrate for the polymerase. By determining differences in labelled acid-insoluble product formed in the reaction it is possible to determine dUTP, dUDP, dTDP and dTTP pools. Ribonucleotide reductase activity was determined by converting either CDP or ADP to its corresponding deoxyribonucleoside disphosphate and then using the diphosphate assay described for deoxyribonucleoside pools.     

Biosynthesis of 5-(4''5''-dihydroxypentyl) uracil as a nucleoside triphosphate in bacteriophage SP15-infected Bacillus subtilis.

The nucleoside triphosphate of 5-(4',5'-dihydroxypentyl)uracil (DHPU) was detected in the acid-soluble extract from bacteriophage SP15-infected Bacillus subtilis W23. No uracil was found in the DNA of either replicating or mature phage. Labeled thymidine added during phage DNA synthesis was incorporated into phage DNA. The presence of DHPU as a nucleoside triphosphate in the acid-soluble pool and the incorporation of thymidine into phage DNA suggest that both DHPU and thymine are incorporated into SP15 DNA via their nucleoside triphosphates. 5-Fluorodeoxyuridine inhibited biosynthesis of SP15 DNA, and this inhibition was reversed by thymidine, resulting in the synthesis of a DNA containing reduced amounts of fully modified DHPU. It is proposed that 5-fluorodeoxyuridine, or its metabolic product, inhibits a step in the biosynthetic pathway to the nucleoside triphosphate of DHPU.     

Deoxyribonucleotide metabolism in Herpes simplex virus infected HeLa cells.

The effect of Rolly No. 11 strain herpes simplex virus infection of HeLa cells in culture on deoxynucleotide metabolism and the level of various enzymes concerned with the biosynthesis of DNA has been investigated. Of 18 enzyme activities studied, thymidine kinase, DNA polymerase and deoxyribonuclease were markedly augmented, a finding in agreement with previous reports. Deoxycytidine kinase, ribonucleotide reductase, thymidylate kinase and deoxycytidylate deaminase activities, in contrast with previous reports, did not increase; the activities of the other enzymes studied, also did not increase. Whereas most of the radioactivity derived from [14-C] thymidine in the acid-soluble fraction of the uninfected cells was present as deoxythymidine triphosphate, that present in the infected cells was primarily in the form of deoxythymidine monophosphate. Thus, in the infected cell deoxythymidylate kinase is a rate-limiting enzyme in the biosynthesis of deoxythymidine triphosphate. A marked increase in the pools of the four naturally occurring deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP) was found. The rate of formation of the virus-induced enzymes was determined, as were the various nucleoside triphosphate pools and the other phosphorylated derivatives of thymidine; a maximum was reached for all these csmponents between 6 to 8 h post infection. Although an apparent greater synthesis of DNA occurred in the uninefected cells, when the specific activity of the radioactive deoxythymidine triphosphate was taken into account, there was actually a greater rate of DNA synthesis in the infected cells, with the peak at 8 h post infection.     

Evidence Suggestive of Compartmentalization of Deoxyribonucleic Acid-Synthesizing Systems in Freeze-Treated Bacillus subtilis

Freezing of Bacillus subtilis in liquid nitrogen results, upon thawing of the cells, in an enhanced deoxyribonucleoside triphosphate and reduced thymidine (Tdr) incorporation into cellular deoxyribonucleic acid (DNA). The DNA synthesized from thymidine triphosphate (TTP) was made by a "repair"-type system as determined by density transfer experiments. The mono- and diphosphate precursors were also incorporated by a "repair"-type synthesis. When Tdr was used as the radioactive precursor in the assay mixture, the product was only that expected from a semiconservative synthesis. Superlethal ultraviolet light exposure of the freeze-treated cells stimulated incorporation of phosphorylated precursors into DNA. Tdr uptake was greatly reduced by ultraviolet exposure, and only repair synthesis was observed. TTP and Tdr do not compete with one another in this system. The possibility that two DNA synthesizing systems exist in separate, non-mixing cellular compartments is considered.     

Biosynthesis of deoxynucleoside triphosphates,dCTP and dTTP: reaction mechanism and kinetics

The enzyme reaction mechanism and kinetics for biosyntheses of deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) from the corresponding deoxycytidine diphosphate (dCDP) and deoxythymidine diphosphate (dTDP) catalyzed by pyruvate kinase were studied. The kinetic model for the two synthetic reactions was found to follow the Bi–Bi random rapid equilibrium mechanism similar to that of the biosynthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP). Kinetic constants involved in the reactions including the maximum reaction velocity, the Michaelis–Menten constants, and the inhibition constants for dCTP and dTTP biosyntheses were experimentally determined. This enzyme reaction requires Mg²⁺ ion and the optimal Mg²⁺ concentration was also determined. The experimental results showed a good agreement with the simulation results obtained from the kinetic model developed. The kinetics of the four biosynthetic reactions for deoxynucleoside triphosphates (dNTP) including dATP, dGTP, dCTP, and dTTP from the corresponding deoxynucleoside diphosphates (dNDP) including dADP, dGDP, dCDP, and dTDP were analyzed. The results suggest that the binding kinetics of phosphoenolpyruvate (PEP) and pyruvate are similar for all four biosynthetic reactions. The affinity of the dNDP substrates to enzyme is of the same order of magnitude as the corresponding dNTP as inhibitors. The order of reactivity and substrate specificity for dNDP is dADP > dGDP > dCDP > dTDP in the pyruvate kinase (PK) reactions. The results obtained from this study can be applied to bioreactor design and production of dCTP and dTTP for biosynthesis of DNA at a significantly lower cost compared to the currently available chemical method.     

On the impossibility of the incorporation of 5-methylcytosine and its nucleosides into higher plant DNA

No radioactivity was detected in 5-methylcytosine isolated from wheat DNA after incubation of wheat seedlings with 3H-labelled 5-methylcytosine, 5-methylcytidine and 5-methyldeoxycytidine. No label from 3H-5-methylcytosine was found in DNA of seedlings. After incubation of seedlings with 3H-labelled nucleosides of 5-methylcytosine, radioactivity was discovered only in thymine of DNA. Thus 5-methylcytosine and its nucleosides can not be used in plants as direct precursors of 5-methyl cytosine residues in DNA, but nucleosides of 5-methylcytosine may be deaminated to thymidine (or deoxythymidine) and subsequently incorporated into DNA.     

An atypical deoxynucleoside triphosphate inBacillus subtilis infected with bacteriophage SP10c

In mature DNA ofBacillus subtilis phage SP10c, deoxythymidine monophosphate is partially replaced by a hypermodified nucleotide. During the interval of phage replication, infected cells contained greatly reduced levels of deoxythymidine triphosphate. However, an atypical mononucleotide, tentatively identified as 5-hydroxymethyldeoxyuridine triphosphate, was present during the interval of SP10c DNA synthesis. It is proposed that the atypical mononucleotide, and not deoxythymidine triphosphate, is a substrate for SP10c DNA replication.     

Biological Activity of 5-Hydroxymethyluracil and Its Deoxynucleoside in Noninfected and Phageinfected Bacillus subtilis

(14)C-hydroxymethyldeoxyuridine (dHMU) is specifically incorporated into the deoxyribonucleic acid (DNA) of bacteriophage SP8. Incorporation experiments demonstrate that the initiation of phage SP8 DNA synthesis occurs between 12.5 to 15 min after infection. Incorporation into host DNA does not occur. (14)C-dHMU can be used as an analytical tool for screening conditionally lethal phage mutants containing hydroxymethyluracil in their DNA to select those that are defective in DNA synthesis under restrictive conditions. The pyrimidine, (14)C-hydroxymethyluracil (HMU), is not incorporated into bacterial or phage DNA. Neither HMU nor dHMU can replace thymine as a growth requirement for Bacillus subtilis 168 Ind(-) Thy(-). HMU does not inhibit the utilization of thymine. Although dHMU inhibits deoxythymidine utilization, the inhibition is not competitive.     

Simultaneous Rates of Ribonucleic Acid and Deoxyribonucleic Acid Syntheses for Estimating Growth and Cell Division of Aquatic Microbial Communities

A method for measuring rates of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) syntheses using a single radioactive precursor has been devised and tested using bacterial cultures and natural assemblages of marine and freshwater microorganisms. The procedure is based upon the uptake and incorporation of exogenous [³H]adenine into cellular adenosine triphosphate and deoxyadenosine triphosphate pools which serve as the immediate precursors for the adenine incorporated into RNA and DNA, respectively. It is proposed that the DNA/RNA rate ratio is correlated with the specific growth rate of microorganisms and can be used as an index for estimating and comparing the productivities of microbial assemblages in nature. This technique can also be used to detect discontinuous growth and cell division processes which frequently occur in surface plankton populations. The DNA/RNA rate ratios measured in a variety of aquatic ecosystems ranged from 3.3 to 31.8% without significant correlation to total microbial biomass.     

Effects of 2',3'-dideoxythymidine triphosphate on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells

2',3'-Dideoxythymidine triphosphate differentially inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells and unscheduled DNA synthesis in bleomycin-treated permeable cells or in isolated rat liver nuclei. The mode of inhibition of 2',3'-dideoxythymidine triphosphate was competitive with respect to deoxythymidine triphosphate. 2',3'-Dideoxythymidine triphosphate inhibited replicative DNA synthesis with a Ki of 8 microM, whereas unscheduled DNA synthesis was more sensitive, the Ki being 0.5 microM. Referring to the differential sensitivity of DNA polymerases alpha and beta to 2',3'-dideoxythymidine triphosphate and to other related information reported previously, the present results suggested that DNA polymerase alpha is playing a major role in replicative DNA synthesis, and DNA polymerase beta in unscheduled DNA synthesis.     

Synthesis of the Unusual DNA of Bacillus subtilis Bacteriophage SP-15

Cultures of Bacillus subtilis infected with phage SP-15 were examined to investigate the metabolic origin of two of the unique components of the phage DNA: the component responsible for the unusually high buoyant density in CsCl and the unusual pyrimidine, 5-(4', 5'-dihydroxypentyl) uracil (DHPU). Newly synthesized pulse-labeled DNA was light in buoyant density and shifted to the high density of mature phage DNA upon further incubation. Parental DNA was converted to a light-density intermediate form prior to replication. When labeled uracil, thymidine, or DHPU were added to infected cells, it was found that only uracil served as the precursor to DHPU and thymine in phage DNA. Analysis of the bases from hydrolyzed DNA of labeled phage or infected cells indicated that the uracil was incorporated into the DNA as such (presumably via deoxyuridine triphosphate) and later converted to DHPU and thymine at the macromolecular level. The sequence of events after phage infection appeared to be: (i) injection of parental DNA; (ii) conversion of parental DNA to a light form; (iii) DNA replication, yielding light DNA containing uracil; (iv) conversion of uracil to DHPU and thymine; and (v) addition of the heavy component.     

PREFERENTIAL LABELING OF RAT LYMPHOCYTES WITH A RAPID RATE OF TURNOVER BY TRITIATED DEOXYCYTIDINE

Lymphocytes in thymic cortex and germinal centers of lymphoid tissues are labeled intensely with generally labeled tritiated deoxycytidine [G-³H]dCyd whereas they are weakly labeled with methyl tritiated deoxythymidine [methyl-³H]dThd of the same specific activity, not only by single injection but also by an intensive injection schedule. [G-³H]dCyd can be used to label short-lived lymphocytes strongly, although not specifically. The distribution patterns of labeled lymphocytes were different depending on the injection schedules of [G-³H]dCyd. [G-³H]dCyd can be used as a precursor molecule for cytosine and also thymine found in DNA. The ratios of radioactive thymine to cytosine measured biochemically on DNA extracted from radioactive lymphocytes labeled by the various schedules indicate strongly that short- and long-lived lymphocyte populations have different abilities to utilize pyrimidine nucleosides for DNA synthesis.     

Increased stability of nucleic acids containing 7-deaza-guanosine and 7-deaza-adenosine may enable rapid DNA sequencing by matrix-assisted laser desorption mass spectrometry.

The use of matrix-assisted laser desorption mass spectrometry (MALDI-MS) has been suggested as an ultrafast readout of Sanger DNA sequencing ladders in a manner analogous to that used with sequencing gels. Currently, a serious limitation of MALDI-MS for the analysis of DNA results from the tendency for oligonucleotides to undergo facile fragmentation in the gas phase. The present study was undertaken to gain an understanding of the influence of various chemical structural features of purine bases on the stability of oligodeoxynucleotide ions produced by MALDI. The study focused on the stability of model compounds of the type d(TTTTTTTTTTXTTTTTTTTT TTTT), where T designates deoxythymidine and X a purine-containing 2'-deoxynucleotide. A variety of different purine derivatives were chosen as the base in the nucleotide X. The mass spectra of the model compounds containing 7-deaza analogues of guanine and adenine reveal a significantly increased stability compared to the 7-aza analogues under the conditions of MALDI-MS. The previously reported incorporation of the 7-deaza-2'-deoxy-adenosine triphosphate and the 7-deaza-2'-deoxy-guanosine triphosphate into DNA by polymerases suggests their use in a Sanger dideoxy sequencing experiment. The dideoxy termination products with the 7-deaza-purines instead of the 7-aza-purines might be sufficiently stable to allow separation and detection of the sequencing ladder by MALDI-MS. Thus, an ultrafast (seconds) read-out of DNA sequence may become feasible.     

Incorporation of thymine-containing DNA precursors in wild-type and mutant T4-infected plasmolysed cells

Summary T4-infected cells, plasmolysed 15 min after infection, incorporate low concentrations (>20 M) of deoxythymidine (TdR) into DNA at a significantly greater rate than dTMP, dTTP or thymine. At higher concentrations (>40 M), dTMP incorporation rate is high, approaching that of TdR at 200 M. TdR is selectively incorporated at all concentrations tested, and is not inhibited by the other thymine containing DNA precursors. Incorporation of low concentrations of TdR requires the T4-induced thymidine kinase (tk) and is not significantly affected by the presence or absence of T4-induced thymidylate synthetase (td). We show that, in T4-infected plasmolysed cells, exogenously added TdR is preferentially incorporated into short DNA fragments during short pulse times. To explain these and other data a model is proposed in which thymidine plays a modulatory role between leading and lagging strand precursor feeds.Preliminary accounts of these data were presented at the West Coast Phage Meetings, Evergreen State College 1980, 1981     

Discrimination between bromouracil and thymine for uptake into DNA in drm- and dra- mutants of Escherichia coli K12.

The relative efficiency of bromouracil and thymine for uptake into DNA was measured in various thymine-requiring strains of Escherichia coli K12. It was found that: 1. Mutants with genotype thyA- dra- discriminate against bromouracil to much greater extent than do mutants with genotype thyA- drm-. 2. The discrimination in dra-mutants is dependent on thymine concentration, whereas discrimination in drm- mutants is almost independent of thymine concentration. It is suggested that the intracellular level of deoxyribose 5-phosphate affects the efficiency of uptake into DNA of bromouracil relative to thymine.     

Alternative metabolic fates of thymine nucleotides in human cells.

Three types of experiments have been used to study the metabolism of thymine nucleotides by human cells. (1) Cells were labelled continuously with [3H]thymidine and the incorporation of label into DNA compared with the specific radioactivities of pools of individual thymine nucleotides separated by chromatography on polyethylene-imine-cellulose. (2) Cellular thymine nucleotides were labelled with [3H]thymidine at 13 degrees C, followed by incubation at 37 degrees C in unlabelled medium. Incorporation of label into DNA and loss of label from the nucleotide pools were monitored during the 'chase' period at 37 degrees C. (3) The experiments described in (2) above were repeated in the presence of the DNA-synthesis inhibitor cytosine arabinoside, in order to demonstrate more clearly and to quantify degradative pathways for thymine nucleotides. In phytohaemagglutinin-stimulated lymphocytes and in bone-marrow cells, only a proportion (25-60%) of labelled thymine nucleotide was incorporated into DNA, the rest being rapidly degraded and lost from the cell. In contrast, an established cell line (HPB-ALL) from a patient with acute lymphoblastic leukaemia of thymic origin incorporated 100% of its exogenously labelled thymine nucleotides into DNA. These results indicated that alternative metabolic routes are open to thymine nucleotides in human cells. In lymphocytes from patients with megaloblastic anaemia and in normal lymphocytes treated with methotrexate, the utilization of labelled thymine nucleotides for DNA synthesis was more efficient than in controls. These results offer an explanation for the observation of a normal pool of thymidine triphosphate in the cells of patients with untreated megaloblastic anaemia even though the amount of this compound available for DNA synthesis appears to be decreased.