Hydroxyurea-Induced Accumulation of Short Fragments During Polyoma DNA Replication I. Characterization of Fragments

Hydroxyurea treatment of 3T6 mouse fibroblast cells infected with polyoma virus resulted within 15 min in more than a 20-fold reduction of the rate of both viral and cellular DNA synthesis. After the initial rapid inhibition, the rate of DNA synthesis remained essentially constant for at least 2 h. In the inhibited cells viral DNA accumulated as short chains with a sedimentation coefficient of about 4S (hydroxyurea fragments). A variable proportion of these fragments was released from the template strands when the viral DNA was extracted by the Hirt procedure. Reannealing experiments demonstrated that hydroxyurea fragments were polyoma-specific and probably synthesized on both parental strands at the replication forks.     

Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism?

Cell-free DNA synthesis was performed in a lysed cell system from mouse cell cultures. The in vitro reaction was totally inhibited by N-ethylmaleimide but unaffected by hydroxyurea or fluorodeoxyuridine when these compounds were added to the incubation mixture. However, in a preparation obtained from cells which had been blocked by hydroxyurea before lysis, the rate of DNA synthesis was markedly reduced. This effect could not have been caused by the depletion of the precursor pools as all necessary triphosphates were added to the in vitro incubation mixture. Analysis by alkaline density gradients showed that the ligation of primary synthesis products is retarded in hydroxyurea-pretreated lysed cells and that small fragments accumulate. These results suggest that hydroxyurea interferes with the processing of early replication products, preventing the formation of longer intermediates. Its mechanism is either independent from the well-known inhibition of ribonucleoside diphosphate reductase or it may be the result of an as-yet-unknown function of this enzyme in a later step of replication. This observation could help to explain why cells appear to be blocked by hydroxyurea in the early part of the S phase (rather than at the G1/S border proper) and also why DNA repair synthesis is relatively insensitive to the drug.     

Accumulation of short DNA fragments in hydroxyurea treated mouse L-cells

Treatment of L-cells with hydroxyurea markedly inhibits the incorporation of [³H]thymidine into DNA. The ³H incorporation that persists during hydroxyurea inhibition is largely into 7S DNA chains. The labelled fragments can be chased into higher MW DNA, suggesting that they are intermediates in the replication process. This interpretation concurs with that of earlier reports which describe a similar effect of hydroxyurea on the replication of viral DNA.     

The recovery of mammalian cells treated with methyl methanesulfonate, nitrogen mustard or UV light. I. The effect of alkylation products on DNA replication.

CHO cells were synchronized in G1 phase and treated with MMS or HN2. The subsequent rate of DNA replication was found to be reduced in a dose-dependent manner. In addition, 2 X 10(-3 M and 3 X 10(-3) M MMS resulted in a 3--4 h delay prior to the initiation of S phase. If the cells were held for 8 h in hydroxyurea after MMS treatment, no subsequent lag in DNA synthesis was seen after removal of the hydroxyurea. The entry of confluent cells into S phase was found to be delayed 7 h upon trypsinizing and replating. Treatment of these cells with MMS resulted in a reduced rate of DNA replication, but no further delay in its initiation. Repair replication was found to continue at a constant rate for at least 12 h following MMS treatment of cells under all of these conditions. At the concentrations used in these experiments MMS severely inhibited the rate of protein synthesis, but HN2 had little effect. By comparing both the kinetics of repair replication and recovery of protein synthesis with the rate of DNA replication, it was concluded that the initial, severe reduction in rate following MMS treatment was probably due to an inhibition of protein synthesis.     

THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.     

Repair replication of opossum lymphocyte DNA: effect of compounds that bind to DNA

Opossum lymphocytes were used for studies of DNA repair. Several compounds were assessed for their capacity to induce repair. Specially interesting was the fact that some intercalators (proflavin, ICR-170, quinacrine and acridine orange) did induce repair, as determined by [³H]thymidine incorporation in the presence of hydroxyurea, CsCl density gradient centrifugation of bromodeoxyuridine-containing DNA and autoradiographically detected unscheduled DNA synthesis.A comparison of the inhibitory effect of several chemicals on DNA replication and DNA repair was also carried out. In this study, repair synthesis was induced by UV irradiation. For most of the compounds, the concentration necessary to inhibit 50% of DNA replication or DNA repair was similar. The most notable exception was cycloheximide which inhibited replication much more effectively than repair. None of the compounds used in this study was found to specifically inhibit repair synthesis.Inhibition of DNA replication and DNA repair was a general effect exhibited by the compounds which bind to DNA. However, only some of these compounds were able to induce repair. As most of these compounds were mutagens it was concluded that the inhibitory effect could be more relevant to mutagenesis that the repair-induction effect.     

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA.     

Inhibition of DNA synthesis and cell death

The association between DNA synthesis inhibition and cell death in mouse L-cells was investigated using the drug hydroxyurea. This drug produces a preferential labelling of low molecular weight DNA and dose-response studies revealed a correlation between this effect and cytotoxicity. Investigation of the reassociation kinetics of DNA labelled during hydroxyurea inhibition showed an over-replication of middle repetitive sequences, but the concentration dependence of this effect was quite different to that of cytotoxicity.     

Hydroxyurea inhibits degradation of internalized epidermal growth factor in HeLa cells

Because epidermal growth factor stimulates DNA synthesis in cultured cells, five inhibitors of DNA synthesis were tested in HeLa cells to see whether the inhibition of DNA synthesis has any effect on the metabolism of the growth factor. Among these, only hydroxyurea inhibited the degradation of ¹²⁵I-labeled epidermal growth factor strongly. The reversal of hydroxyurea-induced inhibition of DNA synthesis by deoxyribonucleosides did not result in a recovery from the inhibition of the degradation. From these findings, it might be concluded that the inhibitory effect of hydroxyurea on the degradation is distinct from that on DNA synthesis.     

The Cik1/Kar3 motor complex is required for the proper kinetochore-microtubule interaction after stressful DNA replication

In budding yeast Saccharomyces cerevisiae, kinetochores are attached by microtubules during most of the cell cycle, but the duplication of centromeric DNA disassembles kinetochores, which results in a brief dissociation of chromosomes from microtubules. Kinetochore assembly is delayed in the presence of hydroxyurea, a DNA synthesis inhibitor, presumably due to the longer time required for centromeric DNA duplication. Some kinetochore mutants are sensitive to stressful DNA replication as these kinetochore proteins become essential for the establishment of the kinetochore-microtubule interaction after treatment with hydroxyurea. To identify more genes required for the efficient kinetochore-microtubule interaction under stressful DNA replication conditions, we carried out a genome-wide screen for yeast mutants sensitive to hydroxyurea. From this screen, cik1 and kar3 mutants were isolated. Kar3 is the minus-end-directed motor protein; Cik1 binds to Kar3 and is required for its motor function. After exposure to hydroxyurea, cik1 and kar3 mutant cells exhibit normal DNA synthesis kinetics, but they display a significant anaphase entry delay. Our results indicate that cik1 cells exhibit a defect in the establishment of chromosome bipolar attachment in the presence of hydroxyurea. Since Kar3 has been shown to drive the poleward chromosome movement along microtubules, our data support the possibility that this chromosome movement promotes chromosome bipolar attachment after stressful DNA replication.     

Characterization of the replication of Escherichia coli DNA in the absence of protein synthesis: Stable DNA replication

After inhibiting DNA synthesis in Escherichia coli, repeated cycles of chromosome replication can occur in the absence of protein synthesis. This “stable” replication requires the products of all of the known dna genes.Stable replication results from inhibiting DNA synthesis by treatment with naladixic acid, cytosine arabinoside or hydroxyurea; or by placing dnaB, dnaE or dnaG mutants at non-permissive temperatures. It also follows a “shift-up” into rich medium in which RNA and protein are synthesized more rapidly than DNA. Paradoxically, stable replication is induced also by treatment with concentrations of streptolydigin which do not inhibit DNA replication but temporarily and partially inhibit RNA and protein synthesis. During all of these treatments, some protein synthesis must occur.Stable replication is not immediately expressed after a short period of thymine starvation or streptolydigin treatment, but requires a subsequent period of protein synthesis. Once established, however, the stable replication state is permanent and will persist in the absence of protein synthesis or during normal growth.After stable replication has been determined by a period of DNA inhibition, it is possible to inactivate replication by heating dnaA, B, C, E and G temperature-sensitive mutants. However, resynthesis of these gene products in the presence of thymine and at the permissive temperature restores stable replication activity. Since restoration of activity can occur under normal growth conditions which do not induce stable replication, it was concluded that the dnaA, B, C, E and G gene products do not directly determine the stabilized character of the replication fork.A model is presented which attempts to explain the ability of different treatments to induce stable replication.     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Enhancement of repair replication in mammalian cells by hydroxyurea

The effect of hydroxyurea on DNA repair replication has been studied in Chinese hamster ovary cells. Mitotic cells were treated with UV irradiation, methyl methanesulfonate or nitrogen mustard and incuated in the presence of each of the 4 [³H]deoxyribonucleosides plus BrdUrd and FdUrd for 2 h. The amount of repair replication was quantitated on CsCl gradients and similar values were obtained for each nucleoside. In all cases addition of HU during the incubation period increased these values approximately 2-fold. Following MMS treatment, pool sizes for each of the nucleosides were estimated by varying the amount of exogenously supplied nucleoside. They were found to be insensitive to the addition of HU and it is concluded that the increased incorporation of [³H]deoxyribonucleosides in the presence of HU reflects an increased amount of repair replication.     

DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.     

The repair of methyl methanesulphonate-induced lesions in the DNA of a murine lymphoma cell

The introduction of single-strand breaks into the DNA of a murine lymphoma (L5178Y) cell treated in vivo with methyl methanesulphonate (MMS) and the behaviour of these breaks on post-treatment incubation were studied. A large proportion of single-strand breaks present after MMS treatment could be repaired as shown by sedimentation in alkaline sucrose. Two inhibitors of DNA synthesis, hydroxyurea and cytosine arabinoside affected the repair process differently-hydroxyurea had only a small effect while cytosine arabinoside blocked repair and at some doses allowed further degradation of the DNA. It was also found that the level of ‘repair replication’ in the presence of cytosine arabinoside was lower than that found in the presence of hydroxyurea.     

Rearrangements of chromatin structure in newly repaired regions of deoxyribonucleic acid in human cells treated with sodium butyrate or hydroxyurea

The rate and extent of redistribution of repair-incorporated nucleotides within chromatin during very early times (10-45 min) after ultraviolet irradiation were examined in normal human fibroblasts treated with 20 mM sodium butyrate, or 2-10 mM hydroxyurea, and compared to results for untreated cells. Under these conditions, DNA replicative synthesis is reduced to very low levels in each case. However, DNA repair synthesis is stimulated by sodium butyrate and partially inhibited by hydroxyurea. Furthermore, in the sodium butyrate treated cells, the core histones are maximally hyperacetylated. Using methods previously described by us, it was found that treatment with sodium butyrate had little or no effect on either the rate or the extent of redistribution of repair-incorporated nucleotides during this early time interval. On the other hand, there was a 1.7-2.5-fold decrease in the rate of redistribution of these nucleotides in cells treated with hydroxyurea; the extent of redistribution was unchanged in these cells. Since hydroxyurea has been shown to decrease the rate of completion of "repair patches" in mammalian cells, these results indicate that nucleosome rearrangement in newly repaired regions of DNA does not occur until after the final stages of the excision repair process are completed. Furthermore, hyperacetylation of the core histones in a large fraction of the total chromatin prior to DNA damage and repair synthesis does not appear to alter the rate or extent of nucleosome core formation in newly repaired regions of DNA.     

Analysis of the schedule of DNA replication in heat-synchronized Tetrahymena

The temporal schedule of DNA replication in heat-synchronized Tetrahymena was studied by autoradiographic and cytofluorometric methods. It was shown that some cells, which were synchronized by selection of individual dividing cells or by temporary thymidine starvation, incorporated [³H]thymidine into macronuclei in a periodic fashion during the heat-shock treatment. It was concluded that supernumerary S periods occurred while cell division was blocked by high temperature. The proportion of cells which initiated supernumerary S periods was found to be dependent on the duration of the heat-shock treatment and on the cell cycle stage when the first heat shock was applied. Cytofluorometric measurements of Feulgen-stained macronuclei during the heat-shock treatment indicated that the DNA complement of these cells was substantially increased and probably duplicated during the course of each S period. Estimates of DNA content also suggested that the rate of DNA synthesis progressively declined during long heat-shock treatments. These results indicate that the mechanism which brings about heat-induced division synchrony is not an interruption of the process of DNA replication. Further experiments were concerned with the regulation of DNA synthesis during the first synchronized division cycle. It was shown that participation in DNA synthesis at this time increased as more cells were able to conclude the terminal S period during the preceding heat-shock treatment. It is suggested that a discrete period of time is necessary after the completion of DNA synthesis before another round of DNA synthesis can be initiated.     

Repair replication in human cells. Simplified determination utilizing hydroxyurea.

A simplified and shortened procedure has been developed for the determination of repair replication of DNA in cultured mammalian cells. The procedure, using the bromodeoxyuridine density label and a radio-isotopic label has been applied to normal diploid human cells (WI38) and to their SV40 transformants (VA13). After incubation with the repair label the cells are lysed and digested for two hours at 50 degrees C with proteinase K. This digest can then be immediately subjected to alkaline cesium chloride density gradient centrifugation with no need for DNA extraction. Hydroxyurea is used to reduce the level of semi-conservative synthesis that a quantitative determination of repair replication can be accomplished by a single centrifugation. The method is not affected by variation in the effectiveness of the inhibitor although a small amount of semi-conservative synthesis normally occurs in the presence of the drug. The time course of repair replication in WI38 cells is unaffected by the drug. The apparent amount of repair synthesis in ultraviolet irradiated cells is increased 25 to 40% in the presence of hydroxyurea when thymidine is used as tracer. Under certain conditions in which the level of semiconservative synthesis is low (e.g., contact inhibited cells, high ultraviolet doses) the use of hydroxyurea is unnecessary.     

Variations in the length of S-phase related to the time cells are blocked at the G1-S interface

The inhibition of DNA synthesis with hydroxyurea or 5-fluorodeoxyuridine decreases the duration of S-phase of synchronously growing Chinese hamster cultures. — The observed drug effects are discussed in relation to an alteration of programmed DNA replication.