A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication.     

DNA replication in methotrexate-treated human lymphoblasts.

The rate of DNA synthesis in cultures of human lymphoblasts decreased more than 80% within 30 min after the cells were exposed to methotrexate, a potent inhibitor of dihydrofolate reductase. Despite this rapid initial inhibition, DNA continued to be synthesized for at least an additional 6 h. The mode of this subsequent replication appeared to be semiconservative, as indicated by the buoyant density of 5-bromodeoxyuridine-substituted DNA in alkaline CsCl gradients. The growth rates of DNA chains in cells exposed to methotrexate were determined by sedimentation rate analysis in alkaline sucrose gradients. DNA synthesized during 2-min or 10-min pulses with labeled deoxycitidine in the presence of methotrexate had about the same sedimentation coefficient, 35 S, as controls. When methotrexate-treated cultures were pulse-labeled for 10 min and then chased for various times, DNA fragments of about 80 S accumulated. DNA synthesized in the presence of methotrexate was stable and elongated to bulk-size DNA after methotrexate inhibition of growth was removed by addition of thymidine and deoxycytidine. The data suggest that methotrexate reduces the rate of DNA replication by inhibiting chain initiation independently of chain elongation.     

A single‐molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed

The replisome catalyses DNA synthesis at a DNA replication fork. The molecular behaviour of the individual replisomes, and therefore the dynamics of replication fork movements, in growing Escherichia coli cells remains unknown. DNA combing enables a single‐molecule approach to measuring the speed of replication fork progression in cells pulse‐labelled with thymidine analogues. We constructed a new thymidine‐requiring strain, eCOMB (E. coli for combing), that rapidly and sufficiently incorporates the analogues into newly synthesized DNA chains for the DNA‐combing method. In combing experiments with eCOMB, we found the speed of most replication forks in the cells to be within the narrow range of 550–750 nt s^?1 and the average speed to be 653 ± 9 nt s^?1 (± SEM). We also found the average speed of the replication fork to be only 264 ± 9 nt s^?1 in a dnaE173‐eCOMB strain producing a mutant‐type of the replicative DNA polymerase III (Pol III) with a chain elongation rate (300 nt s^?1) much lower than that of the wild‐type Pol III (900 nt s^?1). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.     

Chromosomal replication in Drosophila virilis

DNA fiber autoradiography was used to determine parameters underlying the DNA replication of the eukaryotic chromosome in Drosophila diploid brain cells in organ culture. The average rate of fork movement, estimated from 4 different labelling intervals, is 0.35 μm/min at 25 ° C. Of the tandem arrays 93% show patterns which are compatible with bidirectional replication, 7% show unidirectional replication. The unidirectional mode of replication is interpreted as being a consequence of the experimental schedule (using hot-cold pulse labelling) combined with the occurrence of termination signals. — Some autoradiograms showed the expected two grain tracks of different densities; others showed only a high density track. The latter were most prominent in arrays of short replicons (<10 μm) which correlate with replicating satellite sequences. — The majority of replicons fall into size classes < 100 μm. The frequency distribution is skewed towards larger replicon sizes; it spans 2–238 μm, has a mean of ˉx = 35.6 μm and a median of = 21.0 μm. If the distribution is corrected for supposed satellite replicons, the median increases to = 31.0 μm. — In experiments using warmhot pulse labelling, arrays were scored which must have been a consequence of fixed termination signals. Furthermore, grain tracks diverging from weak labelled centers often have different lengths, indicating that these replicons contain two diverging replicating sections of unequal length. Presented to Professor Dr. Wolfgang Beermann on the occasion of his 60th birthday with my best wishes     

Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs

We demonstrate that cell-free extracts prepared from activated eggs of X. laevis by a method similar to that of Lohka and Masui initiate and complete semiconservative DNA replication of sperm nuclei and plasmid DNA. The efficiency of replication is comparable to that in the intact egg. Under optimal conditions 70%-100% of nuclei, and up to 38% of naked DNA molecules replicate completely. Genuine initiation of replication occurs rather than elongation of preformed primers or priming of irreversibly denatured templates. Rereplication of templates is observed under certain conditions. In addition to replicating DNA, these extracts also assemble nucleus-like structures from naked DNA.     

Cyclin B-Cdk1 Kinase Stimulates ORC- and Cdc6-Independent Steps of Semiconservative Plasmid Replication in Yeast Nuclear Extracts

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G₁-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G₁-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40^SIC1, very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G₁-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40^SIC1, restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.     

Inhibition of replicon initiation by 12-O-tetradecanoylphorbol-13-acetate

To investigate the inhibition of DNA replication by tumor promoters, we incubated HeLa cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10^?8 to 10^?5 g/ml) and quantified DNA synthesis on alkaline sucrose gradients. TPA was found to selectively inhibit replicon initiation without affecting DNA chain elongation in replicons that had already initiated. No inhibition of DNA synthesis was seen when cells were exposed to the nonpromoting derivative of TPA, 4-α-phorbol 12,13-didecanoate. Superoxide dismutase did not prevent the TPA-induced inhibition of initiation.     

Studies on the mechanism of DNA replication in Physarum polycephalum

The synthesis of single-stranded DNA subunits (4 × 10⁷ daltons) in Physarum polycephalum was studied by alkaline sucrose density gradient centrifugation. The results were compared with the synthesis of the double-stranded DNA molecules (2.3 × 10⁸ daltons) which they comprise, as determined from neutral sucrose density gradient centrifugation patterns. Although the initiation of synthesis of most double-stranded DNA molecules takes place relatively early in the S period, synthesis of the subunits within them is initiated throughout at least the first two hours of this period. Similarly, replicating (presumably forked) DNA molecules appear to split into daughter DNA molecules prior to the completion of synthesis of the subunits therein. The average rate of DNA chain elongation within subunits is 0.3 × 10⁶ daltons/minute. It is suggested that alkaline sucrose density gradient centrifugation may be a more sensitive method for determining the time required for the completion of replication than other methods based solely on the incorporation of radioactive DNA precursors into an acid-insoluble product.     

Chromatin replication in isolated nuclei from bovine lymphocytes

DNA replication in isolated nuclei from Concanavalin A-stimulated and resting bovine lymphocytes has been studied. Nuclei from S phase lymphocytes incorporate 4–7 times more (³H)dTTP than nuclei from resting cells. The DNA synthesis was dependent on ATP, Mg²⁺ and all four deoxynucleoside triphosphates and was linear for about 60 min. The newly synthesized DNA is nuclear and DNase-sensitive and is the product of discontinuous and semiconservative replication. After limited digestion with micrococcal nuclease the $\text{in vitro}$ replicated DNA was found to occur in nucleosomes prior to joining of primary DNA pieces. Addition of a protein extract from replicating cells stimulated the DNA synthesizing capacity of nuclei from resting lymphocytes. A preliminary characterization of this extract is given.     

Replicating Units (Replicons) of DNA in Cultured Mammalian Cells

Exponentially growing L5178Y mouse leukemic cells were incubated in the presence of 5′-bromodeoxyuridine (BUdR) for about 4 hr, transferred to the nonBUdR-containing medium for a certain period (t hours), and then pulse-labeled with TdR-³H for 10 min. When DNA isolated from these cells was subjected to CsCl gradient centrifugation, the ³H-activity was found to shift gradually from the heavy BUdR-containing peak to the light nonBUdR-containing peak with increasing time t. The average time required for the complete shift of ³H-activity from the heavy to the light DNA fraction was 2.76 hr. Taking this as the average replicating time and the size of DNA fragments in the present preparation as 1.3 × 10⁷ daltons, the rate of replication was found to be 2.1 nucleotides per strand per replicon per sec. By taking the upper limit of the average replicating time as the S period (7.3 hr), various characteristics of the replicating units, such as the lower and upper limits of average size, the average replicating time, the average number of replicating units, etc., were calculated (see Table I).     

Acceleration of proton exchange between octanol and water by 2,4-dinitrophenol determined by 1H-NMR spectrometry a new model for proton transfer in a hydrophobic environment

The rate of proton transfer between the octanol -OH group and water dissolved in octanol after partition equilibrium was determined by ¹H-NMR spectrometry. The rate was found to depend on the pH of the aqueous phase, being minimal at about pH 11. The uncoupler of oxidative phosphorylation 2,4-dinitrophenol at about 10^?3 M accelerated proton transfer several-fold. Its effect was shown to depend on the concentration of the neutral form of 2,4-dinitrophenol in the octanol phase, irrespective of the pH of the aqueous phase. This effect is suggested to be based on the catalytic action of the phenolic -OH group in 2,4-dinitrophenol. The importance of this effect in the uncoupling action of 2,4-dinitrophenol is discussed.     

Rate of DNA replication fork movement within a single mammalian cell

Autoradiography of replicating DNA molecules isolated from individual human cells shows that the rate of DNA replication fork movement within a single cell varies from 0.2 to 1.2 μm/min with an average value of 0.5 to 0.6 μm/min. These data suggest that replication forks move at substantially different rates in different parts of the genome within a single cell.     

Two membrane sites for DNA synthesis in Pneumococcus.

Summary A DNA membrane fraction extracted from pneumococci can be separated into two subfractions with respect to macromolecular composition and DNA synthesis by centrifugation in a 30–60% w/v neutral sucrose gradient. Each fraction can be rebanded in a sucrose gradient or centrifuged to equilibrium in a CsCl density gradient without altering the ability of the fractions to synthesize DNA. The fast sedimenting (heavy) fraction contains 45% of the DNA, and the bulk of the phospholipid, protein, and RNA. The light fraction contains 50% of the DNA, and lower, but significant amounts of phospholipid, RNA, and protein. Both fractions contain a DNA replication complex consisting of a number of enzymes involved in synthesizing DNA or DNA precursors, as well as RNA polymerase activity. However, the specific activity of DNA polymerase in the light fraction is much greater than that in the heavy fraction. In addition, the following results suggest that the former is concerned primarily with replication of the genome while the latter has characteristics of a repair function for the genome. (1) newly synthesized DNA can be detected within 30 s in the light fraction but not until 4 min in the heavy fraction. (2) an RNA-DNA single-stranded hybrid can be demonstrated during initial stages of DNA synthesis in the light, but not heavy fraction. (3) extensive semiconservative DNA replication occurs in the light fraction, whereas little such replication is detected in the heavy fraction. (4) DNA polymerase activity in the light fraction has several of the characteristics of a polymerase identified by others as being concerned with normal DNA replication, such as inhibition by N-ethylmaleimide, and relatively high rates of chain elongation (4.9×10⁴ nucleotides/min). In contrast, DNA polymerase activity in the heavy fraction has characteristic properties associated with DNA polymerase I, a possible repair enzyme. These include higher activity for a d(A-T)n template than that detected in the light fraction, no effect of N-ethylmaleimide, and relatively low rates of chain elongation (9×10³ nucleotides/min).     

Characteristics of an adenosine triphosphatase in erythrocyte membranes stimulated by 2,4-dinitrophenol

Studies were made of the stimulation by 2,4-dinitrophenol (DNP) of an adenosine triphosphatase (ATPase) in stromata of human erythrocytes. Activation by 2,4-dinitrophenol occurs in the range 10^?5 to 10^?3 M and was seen in whole cells, ghosts reconstituted with Mg and ATP, and in osmotic ghosts prepared at a low ratio of cells to water. Phloretin and phloridzin also activated the DNP sensitive system but inhibited it at higher concentrations. DNP increased the K_m and V_max values of the enzyme equally. The DNP sensitive and Na⁺ + K⁺ sensitive enzymes of the stromata were compared. The activities of the two ATPases are additive, require the presence of Mg⁺⁺ and require that the substrate be located at the inner surface of the membrane. The two enzymes differ in their substrate specificity, in their sensitivity to inhibition by ouabain and phloretin and in their sensitivity to some factor in hemolysates. The possible roles of this system in the erythrocyte were discussed.     

Opposite effects of synthetic auxins, 2,4-dichlorophenoxyacetic acid and 1-naphthalene acetic acid on growth of true ginseng cell culture and synthesis of ginsenosides

Effects of synthetic auxins (2,4-D and NAA) on growth of true ginseng (Panax ginseng C.A. Mey) suspension culture and ginsenoside synthesis were investigated. Cell suspensions were grown for 6–8 subcultures on media supplemented with various phytohormones. In all media supplemented with 2,4-D and cytokinins (benzyladenine or kinetin), the cell culture showed sustained growth both in the presence and absence of casein hydrolysate. The average growth index, determined from fresh weight increment over one subculture, equaled to 5.16 ± 0.90, and the maximum mitotic index was 2%. These cell populations having cell volume of 10–17 × 10⁴ μm³ were composed mostly (up to 60–80%) of 5-to 10-cell aggregates with unimodal distribution of nuclear DNA. These cell suspensions were suitable for isolation of protoplasts. The total average content of ginsenosides in the cell culture grown in the presence of 2,4-D constituted 0.18% of dry matter. In media supplemented with NAA, the cell growth was retarded irrespective of the cytokinin species and presence or absence of casein hydrolysate. The growth index (the ratio of final to initial fresh weights) was on average 2.15 ± 0.37, and the mitotic index did not exceed 0.13%. These suspensions, characterized by cell volume of 22–50 × 10⁴ μm³, were composed of large aggregates (> 50 cells). The attempts to isolate protoplasts from these suspensions were unsuccessful. About 25% of cells cultured in the presence of NAA had doubled nuclear DNA content by the end of the subculture. The total content of ginsenosides in cell cultures grown with NAA was on average 4.46% of cell dry matter. The results indicate that ginsenoside synthesis depends on the extent of differentiation in the population of true ginseng cells grown in suspension culture. A certain extent of cytodifferentiation in the cell culture was observed in the presence of NAA, whereas 2,4-D supported only cell proliferation in vitro.     

The segregation of kinetoplast DNA networks in Trypanosoma brucei

The kinetoplast DNA of Trypanosoma brucei consists of 10⁴ minicircles (0.3 μm) and 10² maxicircles (6 μm) held together by catenation in a complex network. In electron micrographs of kinetoplast DNA spread in a protein monolayer we have identified four types of network with the appearance of different stages in network replication and segregation. We show that each network type has characteristic properties with respect to shape, size, number, and location of maxicircle loops and nicked or covalently closed character of minicircles and maxicircles. We propose a detailed model for network segregation that involves a gradual elongation of the network, followed by network cleavage. During this process the basic network structure remains unaltered, implying a complicated mechanism of minicircle rearrangements.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

Eukaryotic error-prone DNA polymerases: The presumed roles in replication, repair, and mutagenesis

A number of error-prone DNA polymerases have been found in various eukaryotes, ranging from yeasts to mammals, including humans. According to partial homology of the primary structure, they are grouped into families B, X, and Y. These enzymes display a high infidelity on an intact DNA template, but they are accurate on a damaged template. Error-prone DNA polymerases are characterized by probabilities of base substitution or frameshift mutations ranging from 10^?3 to 7.5 · 10^?1 in an intact DNA, whereas the spontaneous mutagenesis rate per replicated nucleotide varies between 10^?10 and 10^?12. Low-fidelity polymerases are terminal deoxynucleotidyl transferase (TdT) and DNA polymerases β, ζ, κ, η, ι, λ, μ, and Rev1. The main characteristics of these enzymes are reviewed. None of them exhibits proofreading 3′ → 5′ exonuclease (PE) activity. The specialization of these polymerases consists in their capacity for synthesizing opposite DNA lesions (not eliminated by the numerous repair systems), which is explained by the flexibility of their active centers or a limited ability to express TdT activity. Classic DNA polymerases α, δ, ε, and γ cannot elongate primers with mismatched nucleotides at the 3′-end (which leads to replication block), whereas some specialized polymerases can catalyze this elongation. This is accompanied by overcoming the replication block, often at the expense of an increased mutagenesis rate. How can a cell exist under the conditions of this high infidelity of many DNA polymerase activities? Not all tissues of the body contain a complete set of low-fidelity DNA polymerases, although some of these enzymes are vitally important. In addition, cells “should not allow” error-prone DNA polymerases to work on undamaged DNA. After a lesion on the DNA template is bypassed, the cell should switch over from DNA synthesis catalyzed by specialized polymerases to the synthesis catalyzed by relatively high-fidelity DNA polymerases δ and ? (with an error frequency of 10^?5 to 10^?6) as soon as possible. This is done by forming complexes of polymerase δ or ? with proliferating cell nuclear antigen (PCNA) and replication factors RP-A and RF-C. These highly processive complexes show a greater affinity to correct primers than specialized DNA polymerases do. The fact that specialized DNA polymerases are distributive or weakly processive favors the switching. The fidelity of these polymerases is increased by the PE function of DNA polymerases δ and ε, as well as autonomous 3′ → 5′ exonucleases, which are widespread over the entire phylogenetic tree of eukaryotes. The exonuclease correction decelerates replication in the presence of lesions in the DNA template but increases its fidelity, which decreases the probability of mutagenesis and carcinogenesis.     

ORGANIC MERCURIAL ENCEPHALOPATHY: IN VIVO AND IN VITRO EFFECTS OF METHYL MERCURY ON SYNAPTOSOMAL RESPIRATION

—A reproducible model of subacute methyl mercury (MeHg) intoxication was developed in the adult rat following the daily intragastric administration of 10 mg methyl mercury/kg body wt. Synaptosomes isolated from animals during the latent phase of mercury neurotoxicity (6-10 days) demonstrated no significant change in respiratory control, State 3, State 4, or 2,4-dinitrophenol stimulated respiration with succinate, glutamate or pyruvate plus malate. During the neurotoxic phase, a significant decline in respiratory control was evident with all substrates. Cerebellar synaptosomes revealed qualitatively similar but quantitatively greater inhibition of 2,4-dinitrophenol stimulated respiration during the latent and neurotoxic phases with glutamate. In vitro studies of synaptosome respiration, oxidative phosphorylation and respiratory control with 5-15 μm -methyl mercury revealed a stimulation of initial State 4 respiration, loss of RCI, inhibition of State 3 but no change in the gramicidin or 2,4-dinitrophenol uncoupled rate supported by pyruvate-malate. Phosphate did not relieve the State 3 inhibition. At 25 μm -methyl mercury and above, considerable inhibition of electron transfer occurred. At this concentration, cytochrome c oxidase was inhibited 50%. Isosmotic replacement of medium KC1 by mannitol reduced the MeHg stimulation of State 4 respiration but had no effect on MeHg inhibition of ADP stimulated respiration. Half-maximal stimulation of State 4 respiration by MeHg occurred at [K]⁺⋍ 6 mm . These findings are compatible with an energy-linked methyl mercury induced cation translocation across the synaptosome (mitochondrial) membrane.