Repair Replication in Escherichia coli As Measured by the Photolysis of Bromodeoxyuridine

The ability to selectively photolyze bromouracil-(BrUra-)containing repaired regions in cellular DNA has allowed us to estimate the average size of repaired regions in ultraviolet (UV) light-irradiated Escherichia coli. Cells were labeled with thymidine-³H, irradiated at 254 nm, and incubated in nonradioactive bromodeoxyuridine (BrdUrd). After incubation the cells were exposed to 10⁶ ergs·mm^-2 at 313 nm, lysed, and sedimented in alkaline sucrose gradients so as to measure the average molecular weight of single DNA strands. In strains that had excised ~45 cyclobutane pyrimidine dimers/10⁸ daltons, the 313 nm treatment resulted in ~6 single-strand breaks/10⁸ daltons. In an excisionless strain, the same treatment resulted in only 1.5 breaks/10⁸ daltons. From the determination of the sensitivities of fully substituted DNAs to 313 nm light, we calculate that the repaired regions in excising strains of E. coli contain an average of 4-6 BrUra residues. Photoreactivation experiments indicate that the excision of pyrimidine dimers in the presence of BrdUrd is the primary source of repaired regions selectively photolyzed by 313 nm radiation.     

Effect of Caffeine on DNA Synthesis in Mammalian Cells

Alkaline sucrose sedimentation studies of DNA from mouse lymphoma cells (L5178Y) treated with caffeine have demonstrated the following effects. Caffeine (at a concentration of 1.6 mM) does not introduce strand breaks into preformed DNA nor does it inhibit the rejoining of γ-ray-induced strand breaks. Although it does not affect the over-all rate of DNA synthesis, pulse labeling experiments show that the DNA strands synthesized in its presence are smaller than those made in its absence. This could be the result of (a) DNA being made in shorter replicating units or (b) small gaps in the daughter DNA strands within normal-sized replicating units. These two alternative models were indirectly distinguished as follows. After a pulse label with thymidine-³H in the presence of caffeine, cells were incubated without caffeine in bromodeoxyuridine (BrdUrd). During this incubation, growing strands are elongated and hypothetical gaps (model b) filled in with bromuracil (BrUra)-substituted DNA. The BrUra-containing DNA segments will now be of different lengths on the two models. With smaller replicating units (a) the “elongation segments” will be somewhat smaller than but the same order of magnitude as those in untreated cells, whereas with small gaps (b) the “filled-in gap segments” would be expected to be at least an order of magnitude smaller. The BrUra-containing regions of DNA can be selectively broken open by exposing the cells to light at 313 nm. The exposure required to break open the BUra-substituted regions is inversely related to, and hence gives a measure of, the size of these regions. In caffeine-treated cells these regions were found to be somewhat smaller than but of comparable size with those in untreated cells; this is consistent with the DNA being synthesized in smaller units and argues against the presence of small gaps in the daughter strands.     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

Effect of 5-fluoro-2'-deoxyuridine (FdUrd) on 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by the BrdUrd/Hoechst quenching effect

The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells), BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed to BrdUrd for either 20 min, 8 h, or 24 h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (H) technique. Counterstaining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescence was measured in both techniques with a two-parameter flow cytometer, the histograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti-BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 microM or less are applied.     

A UV-sensitive human clonal cell line,RSa, which has low repair activity

The repair activity of a human transformed cell line, RSa, which was found to be highly sensitive to the lethal effects of 254 nm far-ultraviolet radiation, was compared with that of HeLa cells by evaluating the range of UV-induced incorporation of [methyl-³H]thymidine ([³H]dThd) or 5-[6-³H]bromodeoxyuridine ([³H]BrdUrd) into deoxyribonucleic acid. Direct scintillation counting was used for measuring the extent of unscheduled DNA synthesis (UDS) in UV-irradiated cells, which were treated with hydroxyurea or with arginine deprivation. More quantitative measurements were made by using the density labeling and equilibrium centrifugation method for assaying repair replication. All the amounts of UDS and repair replication in RSa cells were markedly below those in HeLa cells. The possible relationships of the low repair activity to abnormally high UV sensitivity in RSa cells are discussed.     

Differential giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography

Chinese hamster ovary cells grown for two rounds of DNA replication in the presence of BrdUrd contain sister chromatids that fluoresce differentially when stained with Hoechst 33258. If such fluorescent treatments are followed by incubation in 2 X SSC or water at 62° C and staining in 3% Giemsa, the chromosomes now contain one dark (unifilarly substituted) chromatid and one light (bifilarly substituted) chromatid, i.e. are harlequinized. These preparations do not fade and can be studied without resorting to fluorescence microscopy. Sister chromatid exchanges (SCE's) are seen with great clarity and resolution; and all the chromosomes in a cell can be scored, which is contrary to the usual experience with autoradiography. It was found that a) the yield of SCE's is dependent upon the concentration of BrdUrd in which the cells are grown and that the maximum number of SCE's that can occur spontaneously is 0.15 per chromosome per division cycle, b) the yield of SCE's doubles if the cells are exposed to visible light that can cause the photolysis of BrdUrd-containing DNA, and c) chromosomes that appear isolabelled in autoradiographic preparations come from observable multiple exchanges and are not the result of the segregation of DNA from a binemic chromosome. Furthermore, the staining patterns obtained in endoreduplicated cells clearly confirm that the polynucleotide strands of the DNA segregate into sister chromatids as though the newly synthesized strands were laid on the outside of the replicating double helix.     

The mechanism of bilirubin-photosensitized DNA strand breakage in human cells exposed to phototherapy light

Exposure of normal human fibroblasts to visible light (420–490 nm) in the presence of exogenously added 1–100 μg/ml bilirubin enhanced the level of DNA strand breakage compared with cells irradiated in the absence of added bilirubin. Treatment of cells in the dark with an irradiated bilirubin solution also induced DNA strand breaks. However, strand breakage was not detected in cells treated with an irradiated bilirubin solution that had been incubated with catalase (H₂O₂: H₂O₂ oxidoreductase EC 1.11.1.6). Examination of irradiated bilirubin solutions demonstrated the presence of hydrogen peroxide although, apparently, not at concentrations sufficient to account for the level of DNA strand breakage detected. Hence, irradiation of bilirubin results in the generation of hydrogen peroxide and possibly other peroxides that can cause DNA damage.     

Mutation fixation in MNNG-treated Haemophilus influenzae as determined by transformation

A transformation assay has been used to follow the fixation of mutations to novobiocin resistance induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in Haemophilus influenzae. Very few mutations are produced by recently treated DNA, but many are produced by the DNA from cells that have been incubated for a time after exposure to MNNG. The time course of this mutation fixation is shown to coincide reasonably well with the time course of semiconservative DNA synthesis, as judged by uptake studies and by isopycnic centrifugation of density-labeled cells. Incubation with bromodeoxyuridine (BrdUrd) during the fixation period decreases the number of mutations that are fixed, showing in another way the importance of DNA synthesis for fixation.Mutations fixed in the presence of BrdUrd are not more sensitive to 313-nm radiation than those fixed in its absence, suggesting that these residual mutations are fixed in the absence of extensive DNA replication. Mutations newly fixed in the absence of BrdUrd are much more sensitive to 313-nm radiation than are the same mutations some cell generations later. This shows that the newly fixed mutations are in a state that is different from their final form, either because they are in regions of DNA with special configurations of the strands or because they are in a region of DNA that is a hybrid between an old, alkylated strand and a new strand with some bases different from normal. The data suggest that it is unlikely that anything like all the mutations that are fixed in H. influenzae arise by direct action of MNNG on the replication fork. Many of the results can be explained in terms of fixation during semiconservative replication of premutational lesions, some of which are initially located some distance from the replication fork. The final yield would then depend on the relative rates of removal of the lesions by repair and of fixation by replication.     

Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA.

Incubation of human lymphoid cells with bromodeoxyuridine (BrdUrd) for short periods produces three classes of DNA containing analog: DNAHL (hybrid DNA, density approximately equal to 1.75 g/cm3), DNAint (intermediate density DNA, density approximately equal to 1.71 g/cm3), and DNAHH (DNA with both strands containing analog, density approximately equal to 1.80 g/cm3). Preparations of DNAint yield DNAHH after extensive shearing and/or treatment with single strand specific endonuclease. Cross-linking of pulse-labeled (BrdUrd + 3HdT) DNA in cells by treatment with trioxsalen and near UV light before lysis prevents the appearance of DNAHH.Cross-linking after lysis has little effect. A large fraction of DNAHH is obtained after incubation of cells with caffeine. Extraction of DNA at high salt concentration or cross-linking with trioxsalen and near UV light drastically reduced the amount of DNAHH obtained from caffeine-treated cells. We conclude that most DNAHH arises from in vitro branch migration in isolated DNA growing points.     

Isolation of replicating DNA segments from Chinese hamster cells by density equilibrium centrifugation

When DNA is extracted from Chinese hamster cells grown in the presence of 5-bromodeoxyuridine from the beginning of the S (synthesis) phase until the middle of the first replication round, a significant fraction of total replicated DNA bands at intermediate densities between light-light and light-heavy DNA, in a CsCl gradient. Incomplete bromodeoxyuridine substitution compared with light-heavy DNA justifies the displaced banding of these molecules. Since “intermediate DNA” following alkaline or thermal denaturation gives rise to unsubstituted and fully substituted single strands, its particular density in neutral gradients cannot be ascribed to a uniformly reduced degree of bromodeoxyuridine substitution nor to covalently joined light and heavy strands. The segregation of DNA of intermediate densities into light-light and light-heavy components after shearing suggests that it includes at least one junction between replicated and still unreplicated segments, i. e. one replication fork that may or may not have lost one of its prongs. DNA of intermediate density specifically contains one to two sites sensitive to breakage by Neurospora crassa endonuclease.When a two-minute pulse of tritiated bromodeoxyuridine is given during replication in unlabelled heavy medium, the DNA fragments (mol. wt 35 × 10⁶) containing labelled segments band essentially at intermediate positions and are progressively converted to light-heavy molecules, with increasing duration of chase. The half-life of this pulse-labelled intermediate DNA (about 25 min) is consistent with the proportion of total replicated DNA found at displaced densities (10 to 15%) and, together with the distribution of the intermediate radioactivity, is compatible with the existence of adjacent growing replicons.If DNA is labelled and extracted during the second replication round in the presence of bromodeoxyuridine, “intermediate DNA” with similar properties is found between the light-heavy and heavy-heavy peaks.     

Replicon sizes in mammalian cells as estimated by an x-ray plus bromodeoxyuridine photolysis method.

A new method is described for estimating replicon sizes in mammalian cells. Cultures were pulse labeled with [3H]thymidine ([3H]TdR) and bromodeoxyuridine (BrdUrd) for up to 1 h. The lengths of the resulting labeled regions of DNA, Lobs, were estimated by a technique wherein the change in molecular weight of nascent DNA strands, induced by 313 nm light, is measured by velocity sedimentation in alkaline sucrose gradients. If cells are exposed to 1,000 rads of X-rays immediately before pulse labeling, initiation of replicon operation is blocked, although chain elongation proceeds almost normally. Under these conditions Lobs continues to increase only until operating replicons have completed their replication. This value for Lobs then remains constant as long as the block to initiation remains and represents an estimate for the average size of replicons operating in the cells before X-irradiation. For human diploid fibroblasts and human HeLa cells this estimated average size is approximately 17 micron, whereas for Chinese hamster ovary cells, the average replicon size is about 42 micron.     

Postreplication Repair of Ultraviolet Damage to DNA, DNA-Chain Elongation, and Effects of Metabolic Inhibitors in Mouse L Cells

Alkaline sucrose sedimentation studies of DNA from mouse L cells have demonstrated the following effects of several inhibitors of nucleic acid and protein synthesis on postreplication repair of ultraviolet (UV) damage to their DNA. The DNA newly synthesized by a 2 h [³H]thymidine (dThd) label following 254 nm UV irradiation of 20 J/m² is made in smaller segments of the number average mol wt (Mn) of ~10 × 10⁶ than the control of ~40 × 10⁶. The presence of caffeine at a concentration of 2 mM during the labeling of the irradiated cells reduces the Mn value to 5.8 × 10⁶, which is nearly comparable to, but somewhat larger than the expected distance between dimers in parental DNA. Afterwards, such an interrupted DNA made in the irradiated cells is completely repaired to the present maximum Mn value of 40 × 10⁶ in the consecutive 4 h chase in unlabeled dThd. The presence of the nucleic acid inhibitor, either 2 mM hydroxyurea, 50 μM arabinofuranosyl cytosine, 2 mM excess dThd or 5 μg/ml of actinomycin D (AMD) during 2- to 24-h chase periods after a 2 h postirradiation label prevents the repair to various extents, while 2 mM caffeine completely inhibits it. In the unirradiated cells, these agents except excess dThd and caffeine also interfere severely with normal elongation of nascent DNA made by a 3 min pulse label, but do not appreciably induce single chain breaks of either newly synthesized or parental DNA. The inhibition of the repair by AMD suggests that de novo elongation of DNA to close the gaps in new DNA made in the irradiated cells requires at least a template-dependent DNA polymerase. In contrast, 100 μg/ml of cycloheximide allows to complete the gap-filling repair, while it simply reduces the rates of chain growth for the repair and normal replication. Therefore, the similar sensitivity of gap-filling repair and normal replication towards the above inhibitors indicates that a preexisting DNA polymerizing system appears to be responsible and to play a common role without new protein synthesis, as far as the repair at early time after UV is concerned.     

Differential DNase I Sensitivity of the Two Complementary Nucleosomal DNA Strands in Cycloheximide-Treated Ehrlich Ascites Tumor Cells

Abstract

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibrted protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with ¹⁴C-thymidine and the newly synthesized strands - with bromodeoxyu ridine and ³H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15–20% newly replica ted DNA Analysis of the distribution of ¹⁴C-labeled parental DNA fragments complementary to the ³H-nascent strand has shown that the ¹⁴C-labeled fragments prevail in the region of 30–50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5′-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems tobe induced by interactions in the chromatin.     

Recombinational Events between Exogenous Mouse DNA and Newly Synthesized DNA Strands of Chicken Cells in Culture

When chicken cells are cultivated with mouse ³-DNA and BUdR, ³-labelled light segments are found in replicated strands of cellular DNA and heavy segments occur in DNA strands of mouse origin. This suggests that nascent strands of cellular DNA are partly replaced by pieces of mouse DNA and vice versa.     

Attempts to induce mutations in Haemophilus influenzae with the base analogues 5-bromodeoxyuridine and 2-aminopurine.

Attempts were made to induce mutations in Haemophilus influenzae with the base analogues 5-bromodeoxyuridine and 2-aminopurine. These attempts were unsuccessful. Incorporation studies with BrdUrd showed, in agreement with earlier studies on Escherichia coli, that BrdUrd was discriminated against when dThd was also present but was incorporated to essentially the same extent as dThd when only BrdUrd was present. In this latter case, strands fully substituted with BrdUrd was produced, but survival data suggest that bacteria deriving their DNA by replication on such fully substituted templates were inviable. However, bacteria with about 20% of the thymine substituted with bromouracil were usually viable. No mutations could be detected in the descendants of such bacteria. The reasons for this are discussed and it is concluded that in all probability the replication system in species rarely if every treats incorporated bromouracil as anything except a thymine analogue. The alternative possibility, that the negative results are a consequence of the absence of the reclex (SOS) error-prone repair system in this species, is considered much less likely.     

Formation of mengovirus-like particles in cell-free extracts from virus-infected cells

³H-labeled particles with the density of intact mengovirus in CsCl were detected following the incubation of cell-free extracts from mengovirus infected cells with ³H-UTP in a RNA polymerase reaction mixture. The ³H-particles contained complete strands of ³H-labeled 35 S mengovirus RNA. The viral-like particles were found in the region of a sucrose gradient (150–250 S) where viral-specific RNA polymerase activity is detected.     

The effect of single versus double-strand substitution on halogenated pyrimidine-induced radiosensitization and DNA strand breakage in human tumor cells

To better understand the mechanism underlying halogenated pyrimidine-mediated cytotoxicity and radiosensitization in human tumor cells, a study was undertaken to determine the influence of unifilar (one DNA strand) versus bifilar (both DNA strands) substitution of thymidine by the halogenated bases 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd) in HT29 human colon cancer cells. Unifilar labeling was obtained by incubating cells with IdUrd or BrdUrd for one doubling time. Cells were incubated for at least three doublings to approximate bifilar substitution. Only IdUrd caused significant cytotoxicity, which correlated with incorporation into DNA. Both BrdUrd and IdUrd were potent radiosensitizers. Radiosensitization was linearly correlated with incorporation of both bases regardless of the number of strands in which thymidine was substituted. In contrast, the relationship between radiosensitization and DNA double-strand breakage was critically dependent in the case of IdUrd, but not for BrdUrd, on whether substitution was unifilar or bifilar. These findings suggest that incorporation is the best predictor of radiation sensitivity, and that the induction of DNA double-strand breaks alone does not account for radiosensitization mediated by halogenated pyrimidines in these human tumor cells.     

The incorporation of 5-bromodeoxyuridine into DNA of the area opaca vasculosa of chick embryos

The incorporation of 5-bromodeoxyuridine (5-BrdUrd) into DNA of the area opaca vasculosa (AOV) of chick embryos during organ culture was measured. The AOV from blastoderms of the definitive primitive streak stage will not form red cells in the presence of BrdUrd while the AOV of 1–3 somite blastoderms is unaffected by the presence of 5-BrdUrd. About 90% of the original non-density labeled DNA can replicate in the presence of 5-BrdUrd if the tissues come from the younger sensitive embryos, but only 65% of the original DNA will replicate from tissues of older insensitive embryos. Tissues from embryos of both ages replace about 80% of the thymidine by BrdUrd in each newly synthesized strand of DNA; tissues from embryos of both ages will form DNA of hybrid density after one cell generation, and will also form double-heavy DNA after longer periods of culture in the presence of 5-BrdUrd. During recovery from 5-BrdUrd inhibition during a thymidine chase, the density-labeled DNA is replicated so that the new DNA of normal density is formed, but the original heavy 5-BrdUrd containing strands are conserved. It is suggested that inhibition of red cell formation by 5-BrdUrd may occur by incorporation of 5-BrdUrd into DNA of endoderm cells, rather than by acting only directly on red cell precursors.     

Excess thymidine accelerates nascent replicon maturation without affecting the rate of DNA synthesis

The effects of different concentrations of exogenously supplied dThd on DNA replication were investigated in seedlings of Pisum sativum. Nascent DNA was labeled with either [³H]dThd or [³H]dAdo in the presence of 1·10^?6, 1·10^?5 or 1·10^?4 M unlabeled dThd. The rate of DNA synthesis was determined by measuring the kinetics of radioactivity incorporation into trichloroacetic acid-precipitable material and the size of the nascent molecules was investigated using alkaline sucrose gradients. The results obtained showed that high concentrations of exogenously supplied dThd accelerated the joining of completed nascent replicons without affecting the rate of DNA synthesis. This observation strengthens the hypothesis that the dTTP pool size is one of the factors controlling the timing of nascent replicon maturation.     

Estimation of DNA fork displacement rates from isolated mammalian chromosomes

Chromosomes were isolated from Chinese hamster ovary cells that had been pulse-labeled with ³H-thymidine and bromodeoxyuridine and allowed to proceed to metaphase in the presence of colcemid. The chromosomes were then irradiated with 313 nm light and the length of the pulse-labeled DNA segments was estimated by the distribution of ³H-DNA in alkaline sucrose gradients. The average rate of fork displacement, calculated from this measurement, was 0.9 m/min, which was in excellent agreement with measurements from interphase cells. By the same method, hydroxyurea was shown to retard DNA chain growth by 50–60% in both isolated chromosomes and interphase cells.