Overall Rate Of DNA Synthesis Research Articles

Titanium dioxide nanoparticles activate the ATM-Chk2 DNA damage response in human dermal fibroblasts

The use of nanoparticles in consumer products increases their prevalence in the environment and the potential risk to human health. Although recent studies have shown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a more detailed view of the underlying mechanisms of this response needs to be established. Here, the effects of nano-TiO2 on the DNA damage response and DNA replication dynamics were investigated in human dermal fibroblasts. Specifically, the relationship between nano-TiO2 and the DNA damage response pathways regulated by ATM/Chk2 and ATR/Chk1 was examined. The results show increased phosphorylation of H2AX, ATM, and Chk2 after exposure. In addition, nano-TiO2 inhibited the overall rate of DNA synthesis and frequency of replicon initiation events in DNA-combed fibres. Taken together, these results demonstrate that exposure to nano-TiO2 activates the ATM/Chk2 DNA damage response pathway.

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

Visualization of DNA replication in the vertebrate model system DT40 using the DNA fiber technique.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

An Experimental Model for Assessment of Global DNA Repair Capacity

ABSTRACTCellular DNA is constantly subjected to a significant amount of damage resulting from extracellular as well as intracellular processes. There are various mechanisms to repair DNA damage among which nucleotide excision repair (NER) pathway has the highest degree of versatility for recognizing and repairing potentially hazardous DNA modifications. It is difficult, however, to measure the rate of DNA repair as the synthesis of DNA related to repair constitutes only a small fraction of the overall rate of DNA synthesis in the cell. The current paper presents an experimental setup that allows measuring the rate of repair DNA synthesis based on suppression of the replicative DNA synthesis in order to differentiate the repair-associated synthetic activity. The proposed methodology allows for assessment of repair rate of the DNA damage of any type and in any DNA region undergoing repair irrelevant of the structure of the region in question. It could serve as a basis for development of diagnostic methods for assessment of the capacity for global DNA repair.

Early S phase DNA replication: A search for targets of carcinogenesis

Our studies have revealed that replicating DNA is more vulnerable to adduction than is non-replicating DNA. Contrary to our expectations that the vulnerability to neoplastic transformation induced by carcinogens in synchronized cells would parallel the rate of DNA replication, we actually found that the vulnerability was notably increased early in the S phase and more closely paralleled the rate of entry cells into the S phase (the very beginning of S phase) rather than the overall rate of DNA synthesis. From these findings we hypothesized that there were targets for the neoplastic transformation of cells that were among the earliest replicated sequences in the genome. To test that this hypothesis was plausible we investigated the temporal order of DNA replication during the S phase and showed that the order of DNA replication was far more precisely defined than had been recognized previously. The cell synchronization techniques that made those findings possible made it feasible to demonstrate that only a relatively few sites of DNA replication are identifiable in chromosomal bands at the earliest times in the S phase. The same synchronization techniques enabled us to label DNA replicated when populations of cells were very early in S phase and to isolate and clone this DNA. The clonal elements of this library of DNA prepared in this manner have been sequenced and mapped to the human genome. Efforts are in progress to characterize the genes and sequence features associated with these regions. We have utilized methods to identify and characterize origins of DNA replication as a means of locating the earliest replicating part of these early replicating regions. We have identified several new origins of DNA replication that are activated early and late in the S phase but the features of the chromatin at the origin that determines its time of activation remain obscure. In an effort to improve our ability to identify more origins, particularly adjacent origins in genomic regions, we have combined the methods of DNA combing and FISH analysis of combed DNA to search for DNA precursor incorporation patterns characteristic of origins of DNA replication. Preliminary nascent strand abundance studies appear to have proven the existence of two origins of DNA replication predicted from the precursor incorporation studies. We have found that the combed DNA techniques can be combined with precursor incorporation studies and antibodies to sites of DNA damage to address questions of mechanisms of DNA damage and repair. For example these studies have shown recently that DNA damage is not randomly distributed in the genome and that both inhibition of replicon initiation and inhibition of strand elongation are separately distinguishable as components of the S checkpoint function. It is our hope and expectation that these results and the opportunities that they provide for future studies will enable us to identify possible targets for malignant transformation that explain our observation that cells at the start of S phase are vulnerable to the initiation of carcinogenesis.

Checkpoint Regulation of Replication Dynamics in UV-Irradiated Human Cells

At any moment during S phase, regions of genomic DNA are in various stages of replication (i.e. initiation, chain elongation, and termination). These stages may be differentially inhibited after treatment with various carcinogens that damage DNA such as UV. We used visualization of active replication units in combed DNA fibers, in combination with quantitative analyses of the size distributions of nascent DNA, to evaluate the role of S-checkpoint proteins in UV-induced inhibition of DNA replication. When HeLa cells were exposed to a low fluence (1 J/m2) of 254 nm UV light (UVC), new initiation events were severely inhibited (5-6-fold reduction). A larger fluence of UVC (10 J/m2) resulted in stronger inhibition of the overall rate of DNA synthesis without decreasing further the frequency of replicon initiation events. Incubation of HeLa cells with caffeine and knockdown of ATR or Chk1 kinases reversed the UVC-induced inhibition of initiation of new replicons. These findings illustrate the concordance of data derived from different experimental approaches, thus strengthening the evidence that the activation of the intra-S checkpoint by UVC is dependent on the ATR and Chk1 kinases.

Effects of Xenopus laevis Mitochondrial Single-stranded DNA-binding Protein on Primer-Template Binding and 3′→5′ Exonuclease Activity of DNA Polymerase γ

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.

Inhibition of cell proliferation by interferons. 1. Effects on cell division and DNA synthesis in human lymphoblastoid (Daudi) cells.

Treatment of Daudi cells with human lymphoblastoid interferons for up to 5 days progressively inhibits cell proliferation. For the first 3 days cells continue to grow but with prolonged doubling times; subsequently, net proliferation ceases and is accompanied by a loss of cell viability. We have investigated the changes in labelling of DNA with radioactive precursors which occur during the first phase of the response to interferon treatment. We have shown previously [Gewert et al. (1981) Eur. J. Biochem. 116, 487-492] that inhibition of incorporation of [3H]thymidine into DNA can be accounted for by impairment of thymidine transport and thymidine kinase activity. In spite of this inhibition, the total intracellular dTTP pool is larger in interferon-treated than in control cells. Because of these changes it has been necessary to use other methods to determine whether interferon treatment inhibits the overall rate of DNA synthesis. The results of experiments employing (a) moderately high thymidine concentrations or (b) incorporation of radioactivity from deoxynucleoside triphosphates into DNA in detergent-lysed or permeabilised cell systems indicate that there is in fact relatively little inhibition of the overall rate of DNA synthesis in cells exposed to up to 100 units/ml of interferons for at least 48 h. Furthermore, a similar proportion of cells incorporate [3H]thymidine in control and interferon-treated cultures and there is only a small decrease in the number of cells in S phase after interferon treatment, as revealed by fluorescence-activated cell sorting. These results indicate that cell proliferation may be regulated in this system by a mechanism in which there is a loss of coordination between the initiation of DNA synthesis and the subsequent events required for cell division.

Inhibition and recovery of the rate of DNA synthesis in V79 Chinese hamster cells following ultraviolet light irradiation: Variation in the rate of movement of the replication fork

Chinese hamster fibroblasts (V79 cell line) exhibit the phenomenon of recovery of DNA synthesis from the initial inhibition observed after ultraviolet light irradiation, in the absence of significant excision of pyrimidine dimers. In an attempt to determine whether the initial inhibition and subsequent recovery can be accounted for by parallel variations in the rate of movement of the replication fork, the cells were pulse-labeled with radioactive bromodeoxyuridine at different times following irradiation and their DNA centrifuged in neutral CsCl density gradients. When DNA synthesis inhibition was at a maximum, an accumulation of DNA, of density intermediate between hybrid and nionsubstituted DNA, was noticed in the density-distribution profiles. This intermediate-density DNA has been previously shown to correspond to fork structures, and thus it seems that inhibition of DNA synthesis after irradiation is to a great extent caused by the forks pausing at the lesions. Later on, when recovery in the rate of DNA synthesis occurs, the accumulation of intermediate-density DNA is no longer observed. The density distribution of DNA along the gradient can thus provide an estimate of the rate of movement of the replication fork, and the results indicate that most of the variation in the overall rate of DNA synthesis can be accounted for by a parallel variation in the rate of fork movement.

Effects of 3-aminobenzamide on DNA synthesis and cell cycle progression in Chinese hamster ovary cells

3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, is a potent inducer of sister chromatid exchanges (SCEs). Because of the possible relation between SCEs and DNA synthesis, the effects of 3AB on DNA synthesis and cell cycle progression in Chinese hamster ovary (CHO) cells were examined. Unlike all other SCE-inducing agents whose effects on DNA synthesis have been studied, short term exposures (30–120 min) of 3AB did not inhibit the overall rate of DNA synthesis and this result was independent of the amount of bromodeoxyuridine (BrdU) in the DNA. Longer exposure times (>24 h) did result in an extended S phase, but this was not due to an effect on the rate of DNA chain elongation. 3AB also delayed the entry of cells into S phase. The overall cell cycle delay was dose dependent, approaching 9 h after a 54 h exposure to 10 mM 3AB. Earlier reports that 3AB is neither mutagenic nor cytotoxic were confirmed. Thus 3AB acts to increase SCE frequency by a mechanism distinct from that which causes cytotoxicity and mutagenicity, and does not involve any inhibition in the rate of DNA chain growth.

Mode of action of 9-β- d-arabinosyladenine and 1-β- d-arabinosylcytosine on DNA synthesis in human lymphoblasts

The effects of 9-β- d-arabinosyladenine (AraAde), 1-β- d-arabinosylcytosine (AraCyt) and 2′-deoxyadenosine on DNA replication in cultured human lymphoblasts (CCRF-CEM line) were studied by pulse-labeling cells with [ 3H]-thymidine and analyzing the nascent DNA by velocity sedimentation in alkaline sucrose gradients. At doses that reduced the overall rate of DNA synthesis to 50–70% of control values, both AraAde and AraCyt profoundly inhibited the formation of new replicons, with secondary effects on chain elongation contributing to the total inhibition of DNA synthesis. In contrast, the suppression of DNA synthesis by 2′-deoxyadenosine stemmed mainly from an inhibition of chain elongation. These studies also disclosed that about 100 times more AraAde than AraCyt was required to produce a similar inhibition of DNA replication in CCRF-CEM cells. Determination of intracellular concentrations of the nucleoside triphosphates (AraCTP and AraATP) indicated that 90% inhibition of DNA synthesis was achieved at 1.6 and 25 pmol/l · 10 6 cells, respectively. Studies with cell lysates revealed that the replicative machinery in CCRF-CEM cells is more sensitive to AraCTP than to AraATP. This finding contrasts with earlier research, in which the inhibition of purified DNA polymerase by either AraATP of AraCTP yielded essentially the same K i value. The difference in sensitivity of the cell lysate to these arabinonucleotides may reflect either a target enzyme other than DNA polymerase or, more plausibly, some subtle interaction of the polymerase with other components of the replicative process.

Inhibitory effects of high doses of ConA on S phase lymphocytes: ATP pool modification and rapid switch-off on new replicon initiations

When S phase lymphocytes were treated for various times with high doses of ConA, we observed that labelled precursor incorporation into DNA was suppressed. This inhibition is characterized by its rapid onset, its lectin dose dependence and reversibility by α-methyl-mannoside. The uptake of labelled desoxyribonucleoside precursors is not modified by the treatment. The nucleoside kinase activity tested on cellular extracts showed a slight but significant decrease. However, the fact that the specific activity of newly replicated DNA was not modified indicates that the DNA labelling suppression is not a direct consequence of alterations in pathways of labelled DNA precursor synthesis. The available ATP pool in treated cells decreased by 25% after 30 min and near 50% after 1 h. The decrease in DNA labelling observed is related to a decrease in the overall rate of DNA synthesis. From density shift analysis of very large DNA molecules labelled by [ 125I]UdR BUdR (a large part of a cluster of replicons), as well as velocity sedimentation analysis of pulse-chased molecules, we have demonstrated that ( i) the DNA elongation within active replicons is not blocked; ( ii) the rate of assembly of newly replicated DNA fragments (replicons) seems to be unmodified. Consequently, the initiations of adjacent replicons in operating clusters are not affected. However, the number of clusters which start their replication by initiation of new replicons is greatly reduced after 1 h of ConA treatment.

Mechanism of inhibition of DNA synthesis by cycloheximide in [formula omitted] cells

The mechanisms underlying DNA chain elongation were investigated in Balb 3 T3 cells utilizing both intact cells and a whole cell lysate system. Incorporation of labeled deoxynucleoside triphosphates into short (<10 S) chains greatly exceeded that into long (>10 S) chains up to 1 min of incubation with lysate suggesting that chain growth occurs discontinuously along both strands of DNA. Both the kinetics of incorporation and pulse-chase data showed that short chains were true precursors to large molecular weight DNA. Alkaline sucrose sedimentation analysis of DNA synthesized by the lysate system revealed that Okazaki fragments, 5 S in size, matured into 8 S fragments prior to the formation of large molecular weight DNA. Exposure of cells to 25 μM cycloheximide for 1 h reduced protein and DNA synthesis to 5% and 25%, respectively, of control. Prior inhibition of protein synthesis by cycloheximide delayed the maturation of 5 S fragments into 8 S intermediates but did not significantly affect the joining of 8 S pieces to form large molecular weight DNA in vitro. Alkaline sucrose sedimentation profiles of DNA from cycloheximide-treated cells pulse-labeled with [ 3H]thymidine were significantly different from those of control cells indicating altered initiation and termination patterns of DNA replication in the presence of the inhibitor. On the basis of these data and other evidence in literature, it is proposed that a combination of reduced rate of replicon initiation and impaired maturation of 5 S fragments into 8 S intermediates accounts for the observed decreases in the overall rate of DNA synthesis and in the rate of chain elongation in cycloheximide-treated cells.

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [ 3H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [ 3H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [ 3H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis. Using the assay of [ 3H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [ 14C]leucine or [ 14C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis. We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [ 3H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.

Inhibition of mammalian DNA replication by dichlorobenzimidazole riboside

We have investigated the effect of the nucleoside analogue 5,6-dichloro-1-β- d-ribofuranosyl-benzimidazole (DRB) on DNA synthesis in the L-929 line of mouse fibroblasts. Earlier studies have shown that this compound selectively inhibits the synthesis of a major fraction of nuclear heterogeneous RNA (hnRNA). At 60–75 μM, DRB decreases the rate of nuclear hnRNA synthesis by two-thirds and prevents the appearance in the cytoplasm of almost all poly(A)-containing messenger RNA (mRNA). At similar dose levels, DRB inhibits the overall rate of DNA synthesis as measured by thymidine incorporation by only 20% (after correction for decreased thymidine uptake into total cellular material). Analyses of density gradients of BUdR and [ 3H]thymidine-substituted DNA and of autoradiograms of extended fibers of [ 3H]thymidine-labeled DNA confirm this inhibition and suggest that DRB acts on DNA solely by reducing the rate of replication fork movement. The apparent size of replication units is unchanged by drug treatment. DRB also inhibits the transport of thymidine into cells. As determined by kinetic studies of thymidine uptake, transport is inhibited competitively by drug treatment. Several other aspects of thymidine metabolism are not affected by DRB. It does not alter the size of the total dTTP pool, and there is no breakdown of template DNA in DRB-treated cells.

ISOLATION OF A TEMPERATURE-SENSITIVE CELL CYCLE VARIANT OF CHINESE HAMSTER CELLS

A temperature-sensitive cell cycle variant, ts-1 has been isolated from a pseudodiploid Chinese hamster cell line, D6 after mutagenesis with EMS and cytosine arabinoside selection. The ts-1 grows well at the permissive temperature, 34°C, but not at the non-permissive temperature, 40°C, although some fractions of the cells divide once when shift up the temperature to 40°C. The ts-1 was further examined by autoradiography to determine if any functions at a unique stage in the cell cycle is defective at the non-permissive temperature. The studies on the rates of macromolecular synthesis revealed that the overall rate of DNA synthesis, but not RNA and protein synthesis, was affected at the non-permissive temperature. The cessation of DNA synthesis was mainly due to a decrease in the number of cells synthesizing DNA. These results together with studies on asynchronous and mitotically synchronized cells suggest that the ts-1 is a cell cycle variant defective in a function which is active throughout the entire G1 phase and is required for entry to the S phase, but no longer required once cells enter the S phase.

DNA synthesis in mouse embryo fibroblast cells infected with murine cytomegalovirus

The overall rate of DNA synthesis in mouse embryo fibroblast (MEF) cells infected with murine cytomegalovirus (MCMV) decreased immediately after infection, reaching its minimum at 10–12 hr postinfection (PI), and then increased gradually. CsCl equilibrium centrifugation analysis of synthesis of both host and viral DNA in MEF cells infected with MCMV revealed the following: (1) Host cell DNA synthesis was inhibited by more than 95% by 10–12 hr PI. (2) Viral DNA synthesis began at 10–12 hr PI, and by 22–24 hr PI the rate was slightly higher than the rate of host cell DNA synthesis observed at 0–2 hr PI. (3) Viral DNA synthesis occurred without the concurrent synthesis of any significant amount of host cell DNA, indicating that there was preferential viral DNA synthesis. The inhibition of host DNA synthesis occurred when cells were infected with either uv- or heat-inactivated MCMV. This occurred in the absence of both cytopathic effects (CPE) and the synthesis of viral-induced proteins. In these instances, the suppression of host DNA synthesis was observed only during the first 12 hr PI. At 12–14 hr PI, host DNA synthesis began to increase, and finally reached or surpassed the level seen in mock-infected cells. Since the suppression of the host DNA synthesis occurred in the absence of viral-induced protein synthesis, the possibility that the suppression is caused by one or more of the structural proteins of the infecting viral particles is discussed.

Characterization of the viral inhibitory factor (VIF) demonstrable in adenovirus-induced tumor and transformed cells: I. Effect on DNA and viral capsid protein synthesis

The mode of action of the viral inhibitory factor (VIF) demonstrated in homogenates of type 12 adenovirus (Ad12)-induced hamster tumor cells has been further characterized with respect to its effect on synthesis of type 2 adenovirus (Ad2) DNA and capsid proteins. The overall rate of DNA synthesis in infected cells was reduced in the presence of the inhibitor, and analysis by alkaline sucrose gradient centrifugation confirmed that viral DNA synthesis was markedly reduced. It was found, in addition, that the rate of uninfected cell DNA synthesis was reduced by the tumor cell extracts but, because of the complexity of the cell homogenate, it is not known whether the same factor is responsible for inhibition of both viral and cellular DNA synthesis. Virus capsid antigens, as detected by immunofluorescence, were delayed in their appearance in VIF-treated, Ad2-infected cells, but none was completely missing. Acrylamide-gel electrophoresis confirmed that all peptides were synthesized but at a greatly reduced rate. These results are consistent with the observation of reduced viral DNA synthesis and suggest that: (i) VIF acts either on DNA replication itself or at a step in replication prior to DNA synthesis; (ii) whatever the primary site of action, the inhibition is not complete but permits reduced virus replication in all VIF-treated cells, rather than totally eliminating it in some cells.

Postreplication repair of DNA in chick cells: Studies using photoreactivation

During replication of DNA after ultraviolet irradiation, gaps are left in the newly-synthesized DNA strands in both bacterial and animal cells and these gaps are subsequently sealed by a process known as postreplication repair. In order to test whether it is the ultraviolet-induced pyrimidine dimers which are responsible for the production of these daughter-strand gaps in animal cells, we have used chick embryo fibroblasts. In these cells the pyrimidine dimers are photoreactivable, i.e. they can be split by an enzymatic process dependent on visible or near ultraviolet light. Our results indicate that chick cells possess a postreplication repair system similar to that in mammalian cells; gaps are produced in the newly-synthesized strands and then filled in. If the ultravioletirradiated cells are first photoreactivated to remove most of the dimers, the number of daughter-strand gaps produced is much less than without photoreactivation. This suggests that the dimers are indeed responsible for the formation of many of the gaps in the newly-synthesized DNA. Ultraviolet light also inhibits the overall rate of DNA synthesis. This inhibition is, however, only partly overcome by photoreactivation.

Involvement of RNA polymerase in the synthesis of DNA by growing and toluene-treated cells of bacillusbrevis

Summary The incorporation of [3H] thymidine into DNA by growing cultures of bacillus brevis as well as other Bacilli was strongly inhibited by rifampicin and streptolydigin, suggesting a role for RNA polymerase in DNA replication. On the other hand, DNA synthesis in toluene-treated cells of B . brevis , which was dependent on the presence of ATP or other ribonucleoside triphosphates, was unaffected by streptolydigin or rifampicin. However, upon storage of toluene-treated cells at −20°, sensitivity to inhibition by streptolydigin developed progressively (up to 76% inhibition) over a period of 4 weeks, while the overall rate of DNA synthesis remained constant.