dTTP Incorporation Research Articles

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.

Assessment of mutagenic efficiency of two carcinogen-modified nucleosides, 1,N6-ethenodeoxyadenosine and O4-methyldeoxythymidine, using polymerases of varying fidelity.

Terminal deoxynucleotidyl transferase (TdT) was used to prepare copolymers of dA and 1,N6-ethenodeoxyadenosine (epsilon dA). When used as templates for Escherichia coli DNA polymerase I (Pol I) and compared with poly (dA), normal dTTP incorporation was not significantly affected by the presence of 7% epsilon dA. dGTP misincorporation was only slightly increased and occurred about once for every 500 epsilon dA residues. The error-prone polymerase from avian myeloblastosis virus (AMV reverse transcriptase) increased this error rate 5- to 20-fold to a maximum of 1 dG/25 epsilon dA. No dCTP misincorporation was detected with either polymerase. In transcription with E. coli DNA-dependent RNA polymerase, no errors were revealed by nearest neighbor analysis. Poly (dA) treated with chloroacetaldehyde under conditions producing the same proportion of epsilon dA (without the hydrated form) as the synthesized template behaved in the same manner with a similar low level of misincorporation of dG. Such treatment of alternating poly d(A-T) caused structural changes indicative of crosslinks but did not alter its template properties. Increasing the amount of epsilon dA in either synthesized or modified polymers greatly decreased the template activity without increasing the error rate. It is suggested that epsilon dA generally does not prevent dT incorporation but behaves as a bulky lesion which is bypassed. In contrast to the low mutagenic efficiency of epsilon dA, O4-methyldeoxythymidine (m4dT), in copolymers with dA, directed the misincorporation of 1 dG/12 m4dT with Pol I and 1 dG/3 m4dT with reverse transcriptase. Nearest neighbor analysis of transcripts showed the incorporation of 1 dG/12 m4dT. These data are in agreement with the previous reported mutagenicity of m4dT in alternating poly d(A-T, m4T).

Comparison of the effects of differential inhibitors of eukaryotic DNA polymerases on DNA synthesis in isolated rat liver nuclei

1. 1. To investigate the extent to which some commonly used differential inhibitors of isolated eukaryotic DNA polymerases retain their specificity of action in complex DNA-synthesizing cell-free systems a direct comparison was made of the effects they have on [ 3H]dTTP incorporation in isolated rat liver nuclei. 2. 2. Aphidicolin and araCTP inhibited DNA polymerase α but not DNA polymerase β. 3. 3. N-ethylmaleimide inhibited α and β when the activity of the two enzymes depended upon an exogenous primed template. 4. 4. The activity of the chromatin template dependent DNA polymerase β was either directly or indirectly sensitive to the sulfidryl-group-blocking agent. 5. 5. ddTTP did not manifest any selective inhibition of DNA polymerase β. DNA polymerase α showing an equally high sensitivity to the dTTP analog. 6. 6. Isolated regenerating rat liver nuclei do not offer a highly reliable DNA replication system in vitro.

DNA chain growth as a function of age in intact and permeabilized WI-38 and MRC-5 cells

The rate of incorporation of deoxythymidine triphosphate (dTTP) into acid-precipitable material of permeabilized MRC-5 and WI-38 cells as well as the rate of DNA chain growth in both intact and permeabilized cells was examined as a function of cell age. Although both the total rate of dTTP incorporation and the percentage of labeled cells decreased as cultures aged, we could detect no decrease in the rate of DNA chain growth from passages 29 to 53 for MRC-5 and from passages 34 to 50 for WI-38 cells. Since the older passages were in phase III growth and since, in our hands, the WI-38 cells used for this study senesced at passage 51, we conclude that a decrease in the rate of DNA chain growth is not related to in vitro aging.

Retinoic acid-promoted expansion of total cellular ATP pools in 3T3 cells can mediate its stimulatory and growth inhibitory effects.

Exposure of Swiss 3T3 cells to micromolar quantities of beta-all-trans-retinoic acid (RA) results in either inhibition of growth or stimulation of cellular responsiveness to mitogens, depending on the length of treatment. Inhibition of growth is produced by treatment of the cells with RA for at least 48 hours. The total cellular pools of adenosine 5'-triphosphate (ATP) are markedly increased after 48-hour RA treatment and dose dependence studies show a correlation between the expanded ATP pools and the inhibitor effects. The expansion of total cellular ATP pools by retinoic acid occurs throughout the cell cycle and parallels the cell cycle-dependent fluctuations in total cellular ATP pools of untreated cells. Studies of [3H]thymidine incorporation and labeling indices in intact cells and [3H]dTTP incorporation and labeling indices in isolated nuclei of RA-treated and control cultures suggest that cellular acid-soluble nucleotide pools mediate the inhibition of DNA replication in the 48-hour-RA-treated cells. The stimulatory activity of RA for mitogenic responsiveness is demonstrated by treatment of G0/G1-arrested 3T3 cells with micromolar levels of RA for a maximum of 18 hours resulting in the potentiation of phorbol myristate acetate (PMA)-stimulated transition into S phase of the cell cycle. Marked increases in total cellular ATP and UTP pools are produced by 18-hour treatment of G0/G1-arrested cells with RA, before their exposure to PMA.

Release of nucleotide‐cleaving acid phosphatase from carrot cells grown in suspension culture

Carrot (Daucus carota L. cv. Lunga di Amsterdam) cells grown in suspension culture release into the culture medium a phosphatase capable of converting deoxyribo‐ as well as ribonucleoside triphosphates into nucleosides. The enzyme activity requires acidic pH and allows a prompt utilization of phosphorylated DNA precursors as measured by tritiated dTTP incorporation. Such utilization is partially inhibited by inorganic phosphate and completely inhibited by ATP.

Effect of Ehrlich ascites cell chalone on nascent DNA synthesis in isolated nuclei.

Ehrlich Ascites Tumor (EAT) chalone has been shown to inhibit nascent DNA synthesis by inhibiting DNA polymerase alpha and beta (Nakai, 1976), but one of the problems in studying eurkaryotic DNA replication has been the relative impermeability of the cell membrane to precursors and macromolecules; hence, to circumvent this restriction without sacrificing the integrity of the replication process, a broken cell system utilizing nuclei in aqueous media was investigated. Isolated nuclei appear to continue the process of DNA replication that was proceeding in vivo before their isolation and under optimal concitions are able to initiate new synthesis (Fraser & Huberman, 1977). The effects of partially purified EAT chalone on nascent DNA could be studied directly in this nuclear system, which excluded effects of the cell membrane, nucleotide pools and other cytosol elements. A concentration-related inhibition of [3H]thymidine triphosphate ([3H]dTTP) incorporation was noted over a chalone range of 50-200 micrograms/ml. It appears that chalone can inhibit DNA polymerase alpha directly within the nucleus without an intermediate step such as a cell membrane receptor.

An assay system for factors involved in mammalian DNA replication

An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [ 3H]dTTP incorporation, was only 2–5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation). Highly lysed cells were obtained by treatment with 0.1% Brij-58 and 240 mM KCl, while for the preparation of gently lysed cells, 0.01% Brij-58 and 80 mM KCl were used. Incorporation of [ 3H]dTTP reflected DNA synthesis qualitatively similar to that in intact cells. It was semiconservative, and no repair synthesis was detected unless cells were irradiated with ultraviolet light prior to partial lysis. DNA molecules of 4 S were synthesized and converted to DNA of more than 25 S via 6–12-S intermediates. DNA synthesis was restricted to nuclei from cells in S phase, and cell extract did not induce DNA synthesis in nuclei from cells in G 1 phase. Stimulation of DNA synthesis by cell extract was concentration-dependent. Cell extract activity was recovered to more than 50% after (NH 4) 2SO 4 precipitation. Heat-inactivation experiments suggested that cell extract contained at least two factors stimulating DNA replication. This system may, therefore, be used for the purification and characterization of factors participating in DNA replication of mammalian cells.

Regulation of DNA replication in S phase nuclei by ATP and ADP pools

Synchronized 3T6 (mouse fibroblast) ghost monolayers (isolated nuclei) were utilized to study the effects of ATP and ADP levels on DNA replication in vitro. A system yielding discontinuous semiconservative DNA replication (without any detectable repair) in synchronized S phase nuclei has been developed. Lack of initiation of new sites has been observed in isolated S phase 3T6 nuclei without the presence of cytoplasmic material; DNA synthesis is comprised only of elongation at sites where initiation had previously taken place. DNA synthesis in S phase nuclei proceeded optimally at an ATP concentration of 4-5 mM. High ATP levels as well as high ATP/ADP ratios (produced by an ATP-regenerating system at a variety of ATP concentrations) yielded marked inhibition of [(3)H]dTTP incorporation. The cellular and nuclear pools of ATP and ADP in intact synchronized 3T6 cells were accurately determined by high-pressure liquid chromatography. A good correlation with the studies on isolated nuclei has been observed. Whereas total cellular ATP pools increase during the progression of 3T6 cells from G(1) to S phase of the cell cycle, nuclear ATP pools do not increase, and the nuclear ATP/ADP ratios decrease once the cells enter the S phase of their cycle. These experiments suggest that nuclear ATP pools and ATP/ADP ratios act as S phase controls, regulating DNA elongation at sites where its synthesis has previously been initiated by cytoplasmic factors.

Diadenosine 5',5'''-P1,P4-tetraphosphate triggers initiation of in vitro DNA replication in baby hamster kidney cells.

Addition of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) to permeabilized G1-arrested baby hamster kidney cells resulted in the stimulation of DNA synthesis. No stimulation was observed in cells from exponentially growing cultures. The Ap4A-stimulated [3H]dTTP incorporation was inhibited by nalidixic acid, daunomycin, chloroquine diphosphate, EDTA, and N-ethylmaleimide. It was dose-dependent in regard to the amount of permeabilized cells and of Ap4A. Numerous replication eyes were formed in the DNA molecules of stimulated cells. Pulse-chase experiments showed that the synthesis of DNA was discontinuous, resulting in the appearance of approximately 4S Okazaki fragments and their ligation to high molecular weight DNA. These results strongly suggest that Ap4A stimulated the initiation of DNA synthesis in baby hamster kidney cells that had been arrested in G1 by serum deprival.

DNA polymerase from mesophilic and thermophilic bacteria: III. Lack of fidelity in the replication of synthetic polydeoxyribonucleotides by DNA polymerase from Bacillus licheniformis and Bacillus stearothermophilus

1. 1. DNA polymerase from the mesophile Bacillus licheniformis and the thermophile Bacillus stearothermophilus has been used to study the replication of poly(dA-dT) · poly(dA-dT) and poly(dC) · poly(dG) templates at 37, 45, and 55°C. 2. 2. Incorporation of non-complementary deoxyribonucleoside triphosphates (misincorporation) occurred with both enzymes and both templates. Non-specific incorporation (de novo polynucleotide synthesis, random attachment to existing strands, and tritium exchange of nucleotides) accounted for, at most, a small fraction of the total observed misincorporation. The error rates at 37°C for the complete system were as follows: B. licheniformis: dATP, 1 61 ; dCTP, 1 830 ; dGTP, 1 360 ; dTTP, 1 65 ; B. stearothermophilus: dATP, 1 68 ; dCTP, 1 1430 ; dGTP, 1 440 dTTP, 1 67 . For both organisms, the error rate for dCTP and dGTP was independent of incubation temperature; the error rate for dATP and dTTP was 5–50-fold greater than that for dCTP or dGTP and increased significantly from 37 to 55°C. 3. 3. The ratio of dATP to dTTP incorporation with the poly(dA-dT) · poly(dA-dT) template was independent of temperature and close to unity. The ratio of dCTP to dGTP incorporation with the poly(dC) · poly(dG) template decreased from approx. 0.2 to 0.05 for the mesophile and from approx. 0.06 to 0.03 for the thermophile as the temperature increased from 37 to 55°C.

Changes in deoxyribonucleic acid polymerase activities in synthesis of deoxyribonucleic acid during sporulation of Bacillus subtilis.

The deoxyribonucleic acid (DNA) polymerase activities in Bacillus subtilis strains Marburg 168 (thy-trp2) and D22, a DNA polymerase I-deficient mutant, were measured at various stages of sporulation. The DNA polymerase I activity, which had decreased after the exponential growth, began to increase at the early stage of sporulation, reached a maximum and then again decreased. The activity of neither DNA polymerase II nor III was observed to change so drastically as that of DNA polymerase I during sporulation. The incorporation of [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) into Brij 58-treated permeable cells increased during sporulation. The stimulation of [3H]dTTP incorporation into the cells by irradiation with ultraviolet light was also observed to coincide with DNA polymerase I activity. In strain D22 the activities of DNA polymerase II and III were almost constant with time. Neither change of [3H]dTTP incorporation into Brij 58-treated cells nor stimulation of incorporation by irradiation with ultraviolet light was observed.

Replication of T4 DNA in vitro. II. Assay system for and some properties of gene products required for T4 DNA replication.

[3H]dTTP was not incorporated into T4 DNA in the in vitro system for T4 DNA replication when the system was prepared from cells infected with T4 amber mutants defective in DNA replication. [3H]dTTP incorporation was resumed by adding the missing gene product to the defective system. DNA replication by the reconstituted system proceeded by the discontinuous mode of replication, as observed in the wild-type system. By using this in vitro complementation system, molecular weights of gene 41, 43, 44, 45, and 62 products in the active form were roughly estimated as 60,000, 130,000, 130,000, 60,000, and 130,000, respectively. Complex formation between the products of genes 44 and 62 was detected. Other strong interactions between the gene products tested were not detected by glycerol density gradient sedimentation. Interaction of gene products with denatured DNA was analyzed by using a DNA-agarose column, and the results showed that products of genes 32 and 43 had a strong affinity for DNA.

Replication of bacteriophage T4 DNA in vitro. I. Basic properties of the system

A new in vitro system for T4 DNA replication was developed by concentrating cell lysates on cellophane disks. The time course of [3H]dTTP incorporation into DNA by the system was separated into two phases: one was a very rapid incorporation which was terminated within 2 min (phase I reaction), and the other was a slow but continuous incorporation thereafter (phase II reaction). More than half of the phase I reaction product was Escherichia coli DNA, but the phase II reaction was mostly T4 DNA. Phase II reaction required four deoxyribonucleoside triphosphates, ATP, Mg2+, and KCl. 5-Hydroxymethyldeoxycytidine triphosphate was essential for the reaction and not substitutable by dCTP. The presence of KCN or NaN3 in the reaction mixture did not interfere with [3H]dTTP incorporation, but the addition of deoxyribonuclease completely degraded the system. Alkaline sucrose sedimentation analysis of phage II reaction product revealed that phase II reaction proceeded by the discontinuous mode of DNA replication as in vivo. After T4 infection, the activity for phase II reaction appeared in parallel with the activity of T4 phage DNA replication in vivo.

Initiation of replication in chromosomal DNA induced by extracts from proliferating cells.

Addition of an extract prepared from a proliferating cell line to nuclei isolated from resting tissues such as frog liver and spleen resulted in the stimulation of DNA synthesis as assayed by [3H]dTTP incorporation. This stimulated incorporation of [3H]dTTP required ATP and depended on Mg2+ and deoxynucleoside triphosphates. Pulse-chase experiments showed that the synthesis of DNA in this system was discontinuous, resulting in the appearance of approximately 4S fragments and their ligation to yield higher molecular weight DNA. In addition, electron microscopic analysis of the DNA molecules from the reaction mixture showed that the frequency of replication "eyes" in the extract-stimulated reaction was 10-fold higher than that observed in controls. All of these results strongly suggest that the extract stimulated initiation of DNA replication in the chromatin of normally resting cells. Preliminary characterization by dialysis, heating, and enzyme treatments indicated that the activity is associated with one or more proteins of high molecular weight (greater than 50,000). Comparison of the levels of stimulatory activity in extracts from various mammalian and avian sources showed that the activity was present in cells proliferating either in vivo or in tissue culture. In contrast, extracts from normally resting tissues and cells had no activity. The level of activity present did not appear to be directly related to the levels of DNA polymerase. These results suggest a use for this system in studying regulation of the initiation of DNA synthesis and control of the various phases of the cell cycle.

Stimulation of non-conservative DNA synthesis by ultraviolet light in freeze-treated Bacillus subtilis

We have shown that ultraviolet irradiation increases the non-conservative incorporation of dTTP into DNA using cells made permeable to ATP and deoxynucleoside triphosphates by freezing. The role of DNA polymerase I in this incorporation has been studied using N- ethylmaleimide as a selective inhibitor of DNA polymerases II and III. Ultraviolet stimulation of dTTP incorporation requires ATP and is sensitive to N- ethylmaleimide . However, the inhibitor can no longer block the increase in dTTP incorporation if the cells are incubated for 30 min after irradiation before DNA synthesis is allowed. Since N- ethylmaleimide inhibits excision of pyrimidine dimers from the DNA of irradiated cells, it may be that an early step in dimer excision is required for an increase in dTTP incorporation. Because mutants of Bacillus subtilis lacking DNA polymerase I cannot carry out a high rate of dTTP incorporation when assayed in this manner, and because DNA polymerase I is not sensitive to N- ethylmaleimide while polymerases II and III are sensitive, we conclude that DNA polymerase I very likely catalyzes essentially all of the non-conservative DNA synthesis in both unirradiated and irradiated frozen cells.

Effect of inhibiting the cellular synthesis of RNA, DNA and protein on DNA replicative activity of isolated S-phase nuclei

The sensitivity of DNA replication in isolated S-phase HeLa nuclei to inhibitors added in vivo to cells prior to nuclear isolation and to nuclei in vitro has been studied. Whereas the inhibitors hydroxyurea, cycloheximide, 2-mercapto-1-(β-4-pyridethyl)benzimidazole (MPB), and actinomycin D have little or no effect on nuclear [ 3H]dTTP incorporation when added directly to the nuclei, the in vivo treatment of cells with inhibitors of RNA and protein synthesis rapidly depressed DNA replication and DNA replicase activity of the isolated nuclei. The results suggest a requirement for an unstable protein in the maintenance of replicase sites which are established in S-phase nuclei. When RNA and protein synthesis are allowed, DNA replicase activity is maintained during long intervals in which DNA replication is blocked by hydroxyurea.

Increasing activity of germinating Bacillus subtilis spores to incorporate thymidine triphosphate into deoxyribonucleic acid after detergent treatment.

The incorporation of (3)H-labeled thymidine triphosphate ((3)H-dTTP) into deoxyribonucleic acid (DNA) of germinated and then Brij 58-treated Bacillus subtilis spores was measured to study DNA replication activity of cells. The dTTP incorporation rate was very low in dormant spores, gradually increased as germination proceeded, and reached a level of the vegetative cell activity approximately 4 hr after the start of germination. This is in contrast to the DNA polymerase activity in the cell extract which remained at the same level throughout the germination period. The increase of the dTTP incorporation activity was inhibited by chloramphenicol or phenethyl alcohol. When these inhibitors were added after germination had proceeded, the elevated dTTP incorporation activity gradually decreased. Permeability to dTTP of spores germinated in the presence of chloramphenicol and then treated with Brij 58 was confirmed by (i) (3)H-dTTP incorporation into the treated spores following either electron or ultraviolet irradiation and (ii) release of radioactivity from the treated spores containing radioactively labeled DNA after deoxyribonuclease I treatment.

Evidence that thymine is not a normal metabolite in wild-type Bacillus subtilis

Neither thymine nor deoxythymidine triphosphate (dTTP) is incorporated into DNA of prototrophic Bacillus subtilis, although dTTP is a metabolite in DNA synthesis. We have directly shown that lack of dTTP incorporation is due to its exclusion from the cell. On the other hand, thymine freely enters and exits non-requiring cells, but is not utilized in DNA synthesis. We conclude that thymine is not a normal metabolite in prototrophic B. subtilis.