Increase In Polymerase Activity Research Articles

Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase.

Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

B-255 Engineered Reverse Transcriptases and Taq DNA Polymerases Deliver Robust Yield in the Presence of Clinically Relevant Inhibitors

Abstract Objective and Background All biological information is read, written and edited by enzymes. If you improve the enzymes, you improve data quality for precision diagnostics. Reverse transcriptase (RT) and Taq DNA Polymerase (Taq DNAP) are commonly used to detect RNA and/or DNA viruses in patient samples for point of care (POC) diagnostics. However, crude patient samples bring over inhibitors that inhibit these enzymes leading to a decrease in enzymatic activity and an increase in false-negative results in POC testing. In addition, there is a need for faster turn-around time for patient diagnosis which is enabled by engineering more active and sensitive Taq variants that work in fast PCR set-ups. At Watchmaker Genomics, our vision is to overcome hurdles in clinical medicine by engineering enzymes that confer desired phenotypes (i.e., greater activity, thermostability, and inhibitor tolerance) that will enable more sensitive and rapid detection of diseases in clinical medicine. Methods We take a 3-pronged approach to protein engineering: rational design, computational design and directed evolution. The aforementioned engineering approaches were used to rapidly identify and characterize > 20 RT and Taq DNAP variants. First, we deeply characterized each RT variant by measuring processivity via cDNA length and by measuring inhibitor tolerance and thermostability via RT-qPCR. In RT-qPCR, first strand synthesis (FSS) was done at temperatures from 42–65°C in the presence and absence of various clinically relevant inhibitors. For measuring inhibitor tolerance the ΔCt was calculated to determine the level of inhibition (ΔCt = Ct (with inhibitor) - Ct (without inhibitor)). Next, we characterized the inhibitor tolerance of the Taq variants by performing qPCR reactions in the presence of clinically relevant inhibitors. To determine if the Taq variant would be an appropriate fit for fast PCR, the polymerization activity of the Taq DNAP variants were characterized by annealing a single primer to ssDNA and allowing isothermal extension of the primer while monitoring the SYBR green fluorescence increase. Results and Conclusions Reverse transcriptases termed; StellarScript, StellarScript HT and StellarScript HT+ exhibit high processivity at 42°C. StellarScript HT+ exhibited the highest inhibitor tolerance, processivity and thermostability at temperatures up to 65°C. Based on these key attributes, StellarScript HT+ is well suited to give robust yields with clinical samples. Our Taq DNAP variants exhibited a 2.26 fold increase in polymerase activity over native Taq DNAP. Taq variants were identified that efficiently and robustly (i.e., high yield) amplify DNA in the presence of inhibitors when compared to native Taq DNAP. Based on the increased polymerase activity and inhibitor tolerance, WMG Taq variants are well suited to give robust DNA yields with clinical samples. We were successful in engineering RT and Taq DNAP variants that are highly thermostable, active and inhibitor tolerant and thus will enable breakthroughs in existing POC diagnostics.

PB2 mutations D701N and S714R promote adaptation of an influenza H5N1 virus to a mammalian host.

Mutation D701N in the PB2 protein is known to play a prominent role in the adaptation of avian influenza A viruses to mammalian hosts. In contrast, little is known about the nearby mutations S714I and S714R, which have been observed in some avian influenza viruses highly pathogenic for mammals. We have generated recombinant H5N1 viruses with PB2 displaying the avian signature 701D or the mammalian signature 701N and serine, isoleucine, and arginine at position 714 and compared them for polymerase activity and virus growth in avian and mammalian cells, as well as for pathogenicity in mice. Mutation D701N led to an increase in polymerase activity and replication efficiency in mammalian cells and in mouse pathogenicity, and this increase was significantly enhanced when mutation D701N was combined with mutation S714R. Stimulation by mutation S714I was less distinct. These observations indicate that PB2 mutation S714R, in combination with the mammalian signature at position 701, has the potential to promote the adaptation of an H5N1 virus to a mammalian host. Influenza A/H5N1 viruses are avian pathogens that have pandemic potential, since they are spread over large parts of Asia, Africa, and Europe and are occasionally transmitted to humans. It is therefore of high scientific interest to understand the mechanisms that determine the host specificity and pathogenicity of these viruses. It is well known that the PB2 subunit of the viral polymerase is an important host range determinant and that PB2 mutation D701N plays an important role in virus adaptation to mammalian cells. In the present study, we show that mutation S714R is also involved in adaptation and that it cooperates with D701N in exposing a nuclear localization signal that mediates importin-α binding and entry of PB2 into the nucleus, where virus replication and transcription take place.

PB1-F2 Amino Acids Regulate Influenza A Viral Polymerase Activity

In 2001, the 11th influenza A viral protein PB1-F2 was detected and found to be encoded by an alternative open reading frame in the PB1 polymerase gene. PB1-F2 has several unique functions, including roles in promoting apoptosis, increasing inflammation, and regulating viral polymerase activity. This study focused on a single PB1-F2 function: regulation of polymerase activity. We constructed a minigenome system to determine the influence of PB1-F2 amino acid (aa) mutations on polymerase activity. We examined four types of aa mutations: three species-specific aa mutations and one mutation that alters pathogenicity in mice. We discovered that an arginine (R) residue at aa position 29 is highly conserved in avian-derived virus strains. Introducing this mutation into mammalian strain A/WSN/33 (H1N1) led to a marked increase in polymerase activity in mammalian cells.These findings suggest that as PB1-F2 in H5N1 viruses regulates viral polymerase activity, it could be targeted for control of avian influenza infection and drug discovery.

Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments.

The thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) has been truncated to molecular regions essential for polymerase activity. Two truncated forms of the full-length 832 amino acid Taq polymerase have been constructed according to sequence alignments and the known domain structure of the homologous Escherichia coli DNA polymerase I (E.coli pol I): variant delta288 (lacking the N-terminal 288 amino acid portion) and variant delta413 (lacking the N-terminal 413 amino acid portion). Both protein fragments were stable and showed polymerase activity, albeit specific activity and thermostability of the variant delta413 were significantly decreased compared with the full length Taq polymerase. In order to increase the thermostability of the variant delta413, a three-dimensional model of the polymerase domain of Taq polymerase was built by homology with a model of the Klenow fragment of the E.coli pol I based on the available Calpha coordinates. Consequently two variants were designed and constructed using site-directed mutagenesis. The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface by more hydrophilic ones. Compared with the initial protein fragment, both variant enzymes showed an increase in polymerase activity and thermostability. After the completion of this work, X-ray coordinates of the Taq polymerase became available from the protein structure data bank. A comparison between the homology model and the experimental three-dimensional structure proved the quality of the model.

Alterations in the phosphorylation and activity of DNA polymerase α correlate with the change in replicative DNA synthesis as quiescent cells re-enter the cell cycle

The regulation of DNA polymerase alpha was examined in quiescent, human fibroblast cells stimulated to re-enter the cell cycle by subculturing in fresh serum-containing medium. The level of DNA polymerase alpha activity was measured in cell lysates and after specific immunoprecipitation. DNA polymerase alpha activity increased approximately 10-fold during the period of measurement. The activity increase was coincident with an approximately 60-fold increase in thymidine incorporation in the whole cells representing the first S phase. The large increase in polymerase alpha activity was not predominantly the result of synthesis of new polymerase, since the abundance of the enzyme changed less than 2-fold over the measured period. The quantity of [32P]phosphate incorporated into two subunits (180 and 68 kilodaltons) of DNA polymerase alpha increased approximately 10-fold in parallel with the increase in polymerase activity. The specific activity of the cellular ATP pool remained nearly constant over the period of measurement, indicating that the increase in labeling reflects a true increase in incorporation of phosphate. Results from other laboratories indicate that phosphorylation of DNA polymerase alpha increases its catalytic activity. Our results then suggest that the activity increase observed in DNA polymerase alpha when quiescent, human fibroblasts are stimulated to proliferate is largely caused by a phosphorylation-dependent regulatory process.

Cloning and expression of cDNA for human poly(ADP-ribose) polymerase.

cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambda gt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly (ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambda gt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors [pcD-p(ADPR)P; 3.6 kb] was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase as indicated by the following criteria: A 3-fold increase in in vitro activity was noted in extracts from transfected cells compared to mock or pSV2-CAT transfected cells. A 6-fold increase in polymerase activity in pcD-p(ADPR)P transfected cell extracts compared to controls was observed by "activity gel" analysis on gels of electrophoretically separated proteins at 116 kDa. A 10- to 15-fold increase in newly synthesized polymerase was detected by immunoprecipitation of labeled transfected cell extracts. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

Lack of an effect of estrogen on endogenous RNA polymerase II in guinea pig brain

An early action of estrogen in peripheral target tissues is to increase endogenous RNA polymerase II activity which precedes changes in protein synthesis. Previous reports have indicated that estrogen causes a similar increase in polymerase activity in rat brain regions containing high affinity receptors for this hormone. The present series of experiments was designed to test whether a similar action of estrogen on polymerase activity occurs in guinea pig brain, with the expectation being that this enzyme might prove to be a useful marker for the early actions of estrogen. Injections of 25 or 100 μg estradiol-17β in an ethanol-saline vehicle failed to induce significant changes in polymerase II activity in the basal hypothalamus, preoptic area-septum or cortex 1, 2, 6, 13 or 24 h after hormone administration. It is concluded that the modification of RNA polymerase II activity by estrogen is not a good marker for estrogen action in the brain. This may be due to the intrinsic heterogeneity of the tissue resulting possibly in (1) increased activity in some cells and decreased activity in other cells with no net change in overall polymerase activity after estrogen treatment (2) only a small percentage of the cells responding to the steroid with altered polymerase activity with this change not detectable when whole tissue is measured or (3) only a few genes within the cells being altered in transcription by the steroid.

Cerebellar DNA polymerase activity in hypothyroid and malnourished rats.

Neonatal hypothyroidism in rats resulted in a decrease in the rate of cerebellar cell division and a delay in the time when cell division ceased. Thus, by 35 days total cell number, as measured by DNA content, was normal. The prolongation of cell division was reflected by an elevation of DNA polymerase activity that persisted beyond the time enzyme activity dropped to adult levels in either normal animals or animals equally retarded in their growth by malnutrition. This increase in polymerase activity was associated with a sevenfold increase in tritiated thymidine incorporation into DNA at 21 days of age. These findings support the view that the effects of hypothyroidism on the timing of cell proliferation in the cerebellum are not mediated solely by the well-known decrease in food intake that occurs in hypothyroidism and that might be expected to result in some degree of malnutrition.

Cytoplasmic steroid effects on nuclear RNA polymerase activity in canine mammary carcinomas

DNA-dependent RNA polymerase activity has been studied in isolated nuclei from canine mammary tumours. Initial experiments showed high levels of RNase activity in this tissue; accordingly, routine assays were terminated before loss of acid-precipitable radioactivity was evident. RNA polymerase A and B activity in isolated nuclei were shown to be increased by addition of receptor-containing cytosol previously incubated with oestradiol-17β, dihydrotestosterone or R5020. Where no receptor was present, as measured by saturation binding assays and sucrose density gradient analysis, there was no corresponding increase in polymerase activity. The steroid antagonists tamoxifen and cyproterone did not elicit any response even when their corresponding steroids produced a 1- to 2-fold stimulation of polymerase activity. Steroid-induced effects proved to be dose-dependent, with half maximal responses for oestradiol-17β 8 × 10 −8 M, R5020 2 × 10 −6 M and dihydrotestosterone 9 × 10 −6 M.

Control of RNA content of developing human placenta

Human placentae obtained early in pregnancy or at full term were examined for RNA content per cell, RNA polymerase types and activities, and chromatin template availability. The RNA:DNA ratio fell from 0.7 at 15 to 20 weeks to 0.4 at 40 weeks of pregnancy. Since RNase activities were similar at both times, the reduction in RNA content was attributed not to increased degradation, but to reduced synthesis. At both stages of pregnancy, about 55 to 60 per cent of the RNA polymerase activity in isolated placental nuclei was accounted for by RNA polymerase II, as judged by suppression of activity with alpha-amanitin and by separation of the extracted polymerases on DEAE-Sephadex. The relative roles of changes in polymerase activity and template availability were measured in nuclei from 20- and 40-week placentae. Nuclei showed 20 per cent greater polymerase activity in full-term than in early placentae, but the template availability of isolated chromatin for transcription by RNA polymerase II was 70 per cent less at full term. We conclude that the reduced amount of RNA per cell in the full-term placenta is due to reduced template availability that more than offsets the slight increase in polymerase activity.

Characterization of Simian Virus 40 tsA 58 Transcriptional Intermediates at Restrictive Temperatures: Relationship Between DNA Replication and Transcription

When nuclei from simian virus 40 (SV40)-infected cells are lysed with Sarkosyl and the chromatin is pelleted, the supernatant fluid contains a nucleoprotein complex capable of synthesizing viral RNA (Laub and Aloni, Virology 75:346-354, 1976; Gariglio and Mousset, FEBS Lett. 56:149-155, 1975). The level of activity of the RNA polymerase in the complex increased during infection in parallel with the amount of viral DNA that had been synthesized. If cells infected at 33 degrees C with the SV40 mutant tsA 58 were shifted to the nonpermissive temperature of 40 degrees C at any time between 18 and 48 h postinfection, no viral DNA replication was detected after 45 min and no new rounds of synthesis were initiated after 20 to 30 min. However, after this shift, polymerase activity associated with the nucleo-protein complex did continue to increase for 5 h, at which time it reached a plateau. There was an increase of RNA synthesized from both the early (E) and late (L) SV40 DNA strands, and there was a threefold increase in the ratio of early-to-late RNA species after the shift. In comparable experiments with cells infected with wild-type virions, no increase in polymerase activity occurred because of the temperature change alone. At 33 degrees C, the relative amount of RNA transcribed from the wild-type E-strand was less than tsA 58 at 33 degrees C and did not increase after a shift to 40 degrees C. The tsA 58 transcriptional complexes extracted from cells grown at 33 degrees C sedimented heterogeneously in sucrose gradients, with a peak near 26S. There were no detectable alterations in the sedimentation properties of the complexes when tsA 58-infected cells were shifted to 40 degrees C for 2 h. We conclude that continued synthesis of viral DNA is not an obligatory prerequisite for maintenance of late viral transcription nor is the sedimentation of the transcriptional complex at 26S related to actively replicating DNA molecules serving as templates for transcription. Further, an increase in late transcription can occur under conditions where reinitiation of viral DNA synthesis is prevented. The increase in the synthesis of early and late RNA at the restrictive temperature without concurrent DNA synthesis is discussed in relationship to the function of the A gene product.

Phosphorylation of yeast RNA polymerases.

DNA-DEPENDENT RNA polymerase activities in eukaryotic cells change during developmental1,2 and physiological transitions3–8. Some changes occur rapidly and in the absence of protein synthesis. They presumably involve modulation of the activity of pre-existing RNA polymerase molecules rather than alterations of the rate of synthesis or degradation of these molecules. This modulation of polymerase activity could be a consequence of covalent modification of enzyme subunits, the rapid turnover of a protein factor required for catalysis, alteration of the template properties of the chromatin or some combination of these. The first possibility is attractive. The activity of many enzymes is controlled in this manner9. Cycles of phosphorylation and dephosphorylation regulate the activity of the enzymes involved in glycogen metabolism (reviewed in refs 9 and 10). Lee et al.11 observed an increase in the specific activity of Rous sarcoma virus reverse transcriptase on incubation with protein kinase and ATP. Phosphoryla-of β and β′ subunits of E. coli RNA polymerase on infection by bacteriophage T7 has been implicated in the control of early transcription of T7 DNA12. Several groups5,13,14 have proposed and presented preliminary evidence that phosphorylation of RNA polymerase is involved in regulation of polymerase activity in eukaryotes. Jungmann et al.5 and Martelo and Hirsch13 observed enhanced RNA polymerase activity when partially purified protein kinase, RNA polymerase and ATP were mixed. They observed the phosphorylation of proteins in their preparations and attributed the increase in polymerase activity to phosphorylation of the enzyme. They did not demonstrate phosphorylation of RNA polymerase polypeptides, and since the observed increase in RNA polymerase activity could also be a consequence of phosphorylation of contaminating acidic nuclear proteins which can stimulate polymerase activity15–17, the cause of the enhanced polymerase activity is obscure. We report here the isolation of phosphorylated RNA polymerase I from yeast cells and the identification of the enzyme polypeptides phosphorylated in vivo. The same pattern of phosphorylation was obtained when highly purified RNA polymerase I was incubated with a yeast protein kinase preparation.

RNA synthesis and RNA polymerase activity in hepatic nuclei isolated from rats fed the carcinogen 2-acetylaminofluorene

An assessment was made of the activity of RNA polymerase I and the capacity for RNA synthesis, under conditions optimized for RNA polymerase I activity, in hepatic nuclei isolated from rats fed a diet containing the hepatic carcinogen AAF. Animals were maintained on the carcinogenic diet for either 4, 7 or 14 days. RNA polymerase activity progressively increased with time on the carcinogenic diet. However, the capacity for RNA synthesis remained quite constant. These results are suggestive of a progressive inhibition of DNA template activity during the early stages of AAF-induced hepatocarcinogenesis. The “permanance” of the increase in polymerase activity was examined by switching carcinogen fed animals to a control diet for either 2 or 5 days prior to making an assessment of the above parameters.

DNA Polymerases in Normal and Leukemic Human Hematopoietic Cells

Abstract DNA polymerase activities were assayed in bone marrow cells and peripheral leukocytes from normal people and patients with acute myelogenous, chronic lymphocytic, and chronic myelogenous leukemia. Extracts of subcellular components were fractionated by velocity sedimentation through sucrose density gradients and assayed using activated DNA as template. Two major DNA-dependent DNA polymerases were found in human cells with molecular weights of approximately 50,000 and 200,000 daltons, respectively. The DNA polymerase of high molecular weight is located in the soluble cytoplasmic fraction and is inhibited by N-ethylmaleimide. The low molecular weight polymerase is detected in extracts of nuclei and in the soluble fraction. It is resistant to inhibition by N-ethylmaleimide. In all cell types tested, total DNA polymerase activities were much higher in cytoplasmic than in nuclear extracts. Lymphocytes purified from normal peripheral blood had three to four times as much of both the high and low molecular weight polymerase activities per cell as purified granulocytes. Leukemic myeloblasts had 10 to 20 times as much cytoplasmic DNA polymerase activity as more mature leukocytes from normal peripheral blood. In general, immature granulopoietic cells contained higher total DNA polymerase activities than more mature granulocytes, and the major increases in polymerase activities were in the high and low molecular weight cytoplasmic enzymes rather than in the nuclear enzyme.

Changes in Poly(Adenosine Diphosphate Ribose) Polymerase on Stimulation of Pig Lymphocytes with Phytohaemagglutinin

Peripheral mammalian lymphocytes grown in culture have low rates of RNA and protein synthesis and do not synthesize significant amounts of DNA. On addition of the mitogen phytohaemagglutinin, the cells undergo a series of changes including increases in RNA and protein synthesis, and culminating in DNA synthesis and cell division. DNA synthesis commences about 24h after stimulation. The present communication describes changes in the nuclear enzyme, poly(ADP-ribose) polymerase, in phytohaemagglutinin-stimulated pig lymphocytes. This chromatin-bound enzyme converts NAD+ into poly(ADP-ribose) with the elimination of nicotinamide, the product being covalently attached to chromosomal proteins (see, e.g., Nishizuka et af . , 1969). The reaction is absolutely dependent on the presence of DNA. The function of the polymerase and the polymer remain to be elucidated. Poly(ADP-ribose) polymerase activity was measured as the uptake of [3H]NAD+ into acid-insoluble material in isolated nuclei. The activities of the polymerase 24 and 48h after phytohaemagglutinin stimulation were respectively 1.5 and 2.9 times that in unstimulated cells. Thus approximately one-quarter of the increase in activity occurred in the first 24h after stimulation, before the onset of DNA synthesis, the major increase occurring concomitantly with DNA synthesis. The increase in activity of poly(ADP-ribose) polymerase could produce either more poly(ADP-ribose) chains of the same length (i.e. more initiation of new chains) or the same number of longer chains (more elongation). To distinguish between these alternatives, nuclei were incubated with [3H]NAD+and after various digestion procedures the polymer was hydrolysed at the phosphodiester linkages. This gives rise to AMP and 2'-(5-phosphoribosyl)-5'-AMP. The amounts of radioactivity in AMP and phosphoribosyl-AMP were measured after separation on Dowex formate, and the ratio of total radioactivity to radioactivity in AMP gives a measure of the chain length of the polymer (Fujimura & Sugimura, 1971). This length was found to be the same (about eight nucleotides) for unstimulated and 48 h-stimulated cells. Thus the increase in polymerase activity is associated with increased initiation of poly(ADP-ribose) chains, not with longer chains. The effects of culturing the lymphocytes after phytohaemagglutinin stimulation in the presence of various metabolic inhibitors were studied. Although hydroxyurea (an inhibitor of DNA synthesis) partially prevented the increase in poly(ADP-ribose) polymerase activity, fluorodeoxyuridine, at concentrations that completely prevented DNA synthesis, produced only a l0-15% inhibition of the increase in activity seen after 48h. Low doses of actinomycin D (5-25ng/ml), which prevented the increases in DNA and protein synthesis to varying degrees, inhibited the increasein poly(ADP4bose) polymerase activity to similar extents. Thus the stimulation of this activity seems to depend on general macromolecular synthesis, but to be independent of concomitant DNA synthesis.

DNA polymerase activity in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Requirements and optimal conditions have been studied for the activity of DNA polymerase from phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Differences were found in thermal stability and inhibitory effect of KC1 and p-chloromercuribenzoate. The relationship was determined between DNA polymerase activity, cellular pools of dATP, dTTP and incorporation of deoxythymidine into DNA during transformation. The increase in polymerase activity was paralleled by a similar increase in the pools of dATP and dTTP. The enzyme activity and the pool sizes of both nucleotides reached a maximum simultaneously with the peak of deoxythymidine incorporation into DNA. Studies in which protein synthesis was limited by cycloheximide showed that both the DNA polymerase activity and the rise in the pool sizes of both nucleotides were abolished. This implies that the de novo synthesis is required for the enzymes involved.

Fluctuations of DNA-dependent RNA polymerase and synthesis of macromolecules during the growth cycle of Novikoff rat hepatoma cells in suspension culture.

AbstractThe transition of suspension cultures of Novikoff rat hepatoma cells from the exponential to the stationary phase is accompanied by decreases of over 90% in the rates of synthesis of RNA, DNA and protein, a 90% loss of the apparent DNA‐dependent RNA polymerase activity of the cells, and a disaggregation of the polyribosomes with a concomitant accumulation of 80 S and 110 S ribosomal structures. The cells also attain a minimum content of DNA, RNA and protein and a minimum size. Upon dilution of stationary phase cultures with fresh medium, the rate of protein synthesis begins to increase immediately and this correlates with a rapid reformation of the polyribosomes. The initial re‐formation of polyribosomes is little affected by the presence of actinomycin D. RNA polymerase activity also begins to increase immediately after dilution and an increase in rate of RNA synthesis becomes apparent shortly thereafter. The increase in polymerase activity is inhibited by treating the cells with puromycin or actidione. Cell division commences only 9–13 hours after dilution and the rate of DNA synthesis begins to increase about midway through the lag period. During the lag period the average cellular content of protein increases about 80% and that of RNA and DNA about 30%. These increases are accompanied by a marked increase in the average size of the cells.Upon continued incubation of stationary phase cultures, the cells become irreversibly damaged physiologically before gross morphological damage becomes apparent. The irreversible physiological damage is recognized by the fact that the cells fail to recover when suspended in fresh medium.

Deoxycytidylate Synthesis and Entry into the Period of Deoxyribonucleic Acid Replication in Rabbit Kidney Cells

Abstract With kidney cells cultured directly from the animal, increases in the labeling of the deoxycytidylate pool from 3H-cytidine and in deoxyribonucleic acid polymerase activity begin at about the time of initiation of DNA formation. Some of the possible causal interrelationships of these three changes have been examined. With the use of phleomycin to block DNA synthesis, it has been shown that neither the increase in dCMP nor the increase in polymerase activity is triggered by the act of DNA replication. Hydroxyurea reversibly blocks the rise in the labeling of the dCMP pool. With this inhibitor, it has been shown that the normal increase in the deoxyribonucleotide pool is not essential for the increase in DNA polymerase activity. Nor does the rise in the deoxyribonucleotide pool appear to be required for entry of a kidney cell into the period of DNA synthesis. When cells inhibited with hydroxyurea were irradiated with x-rays (which prevent entry into the replicative stage but have little effect upon the continuation of DNA formation), removal of the chemical inhibitor was followed by the synthesis of DNA.