Synthesis of small polynucleotide chains in thymine-depleted bacteria

Bacteria that are depleted of intracellular thymidine nucleotide pools incorporate [³H]thymine at full specific activity, allowing the detection of early intermediates in DNA replication. A short pulse of [³H]thymine is incorporated almost exclusively into very small DNA chains which, during further incorporation of thymine, are converted into larger chains and high molecular weight DNA. The synthesis of these small DNA chains depends on the products of dna genes B, E and G. Analysis of the DNA by gel filtration on Sepharose 2B revealed an abundance of extremely short DNA chains while the frequency of larger chains decreased exponentially with increasing size. This size distribution of small DNA chains suggests a mechanism of DNA replication in which larger chains (Okazaki pieces, Okazaki et al., 1968a) arise through joining of extremely short polynucleotide chains in a process resembling crystallization rather than unidirectional chain elongation.     

Characterization of Simian virus 40 DNA component II during viral DNA replication

Replicating molecules of Simian virus 40 DNA labeled during a short pulse with [³H]thymidine have been fractionated by ultracentrifugation methods and the open circular form (DNA component II) has been characterized. The pulse-labeled DNA component II is a relatively small constituent (1 to 3%) of the pool of replicating molecules. Examination of the circular (18 S) and linear (16 S) strands of DNA component II by alkaline sedimentation and by degradation using exonuclease III of Escherichia coli reveals that the newly synthesized DNA is principally in the linear strand. Cleavage of pulse-labeled DNA component II by an fi⁺, R-factor restriction endonuclease from E. coli demonstrates that the interruption in the pulse-labeled strand is specifically located at the termination point for replication.During a chase period of 20 minutes the amount of DNA component II increases to about 6 to 8% of the total labeled viral DNA. The kinetics of formation of superhelical, DNA component I and disappearance of replicative intermediates are linear during the chase period. After several hours of continuous labeling of replicating viral DNA, the DNA component II pool consists mainly of molecules labeled in both strands with the interruption non-specifically located in either strand. These molecules probably arise by the random introduction of single-strand breaks in newly synthesized DNA component I. During short periods of continuous labeling with [³H]thymidine, the ratio of DNA components I to II increases as a function of the pulse duration. These results support a model for 8V 40 DNA replication in which the open circular form is a precursor of the superhelical form.     

Gaps in DNA synthesized by ultraviolet light-irradiated WI38 human cells.

DNA replication in ultraviolet-irradiated human cells was examined by treatment of the extracted DNA with a single-strand specific endonuclease from Neurospora crassa. WI38 cells were uniformly labeled with 32Pi for two generations before irradiation and then labeled with [3H]thymidine after irradiation. The isolated DNA was sedimented in neutral sucrose gradients after incubation with the endonuclease. The endonuclease treatment had no effect on the sedimentation profiles of either [32P]DNA or [3H]DNA from unirradiated control cultures. The endonuclease treatment also did not significantly alter the profile of [32P]DNA from irradiated cultures but did introduce breaks in the 3H pulse-labeled DNA synthesized after irradiation. These results indicate that DNA synthesis after ultraviolet irradiation proceeds in such fashion that gaps are formed along the newly made strand, leaving regions of single strandness in template DNA. As replication proceeds these gaps disappear and 2 h after irradiation (100-250 ergs/mm2) they are barely detectable by the endonuclease assay.     

Maturation of newly replicated chromatin of simian virus 40 and its host cell

The DNA in replicating simian virus 40 chromatin and cellular chromatin was labeled with short pulses of [³H]thymidine. The structure of pulse-labeled nucleoprotein complexes was studied by micrococcal nuclease digestion. It was found that in both newly replicated viral and cellular chromatin, a structural state appears which is characterized by an increased sensitivity to nuclease and a faster than usual rate of cleavage to DNA fragments of monomeric nucleosome size and smaller. Pulse-chase experiments show that each of these effects requires a characteristic time to disappear in both systems, suggesting the existence of different sub-processes of chromatin maturation. One of these processes, detectable by the reversion of the unusually fast production of subnucleosomal fragments, is delayed in SV40 chromatin replication.     

Chromatin maturation depends on continued DNA-replication

The structure of [3H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [3H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. We conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

Discontinuous DNA replication in developing sea urchin embryos.

Embryos of the sea urchin, Hemicentrotus pulcherrimus, growing synchronously and entering the third “S” phase at 120 min after fertilization, were pulse-labeled with [³H]thymidine for various periods ranging from 15 sec to 20 min, and the size of nascent DNA was analyzed by centrifugation in an alkaline sucrose gradient. It was found that pulse-labeling for 15 sec gave rise to a sedimentation profile with a major radioactivity peak at the position corresponding to a molecular weight of 4 × 10⁴ daltons. One-minute of labeling, however, gave a major radioactive band around the position corresponding to 1.4 × 10⁶ daltons. Upon increasing the labeling time, the radioactivity peaks or bands shifted toward the increasing molecular weights. Finally, most of the radioactive DNA was found to sediment at the bottom when the embryos were exposed to [³H]thymidine for 15 min or longer. The time span of the S phase in the cleavage embryos was about 15 min. The results of pulse and chase experiments also supported the discontinuous mechanism of DNA replication in the cleavage embryos.     

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

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

Incorporation of uracil into the growing strand of adenovirus 12 DNA.

The amount of rapidly labeled short DNA chains in adenovirus 12(Ad12)-infected cells was markedly increased in the presence of either uridine or deoxycytidine which could be converted to dUTP. When the infected cells were labeled with [³H]uridine or [³H] deoxycytidine and the labeled nucleotides in the short DNA chains from the Hirt supernatant were analysed by thin-layer chromatography, approximately 90 or 20% of the label was detected in dUTP. These results suggest that at least a portion of short DNA chains formed during Ad12 DNA replication is derived from an excision-repair mechanism of uracil containing nascent strands.     

In vitro polyoma DNA synthesis: Involvement of RNA in discontinuous chain growth

Short fragments of DNA (5 S) isolated by denaturation from polyoma replicative intermediates pulse-labeled in vitro were shown to have RNA covalently attached by three criteria: (1) such fragments were slightly denser than bulk viral DNA. (2) They could be labeled directly with α-³²P-labeled ribotriphosphates. (3) Alkaline hydrolysis of fragments labeled with α-³²P-labeled deoxynucleoside triphosphates showed ³²P transfer to 3′ ribonucleoside monophosphates. Except for a preference of transfer from dC, the link showed little sequence specificity. The data are compatible with the notion that all short fragments in replicating viral DNA are initiated by an RNA primer. This RNA is maximally 30 bases long and is rather short-lived.     

Application of hypoxia-induced shut down of replicon initiation to the analysis of replication intermediates in Ehrlich ascites cells

Cultured Ehrlich ascites cells were subjected to hypoxic conditions for about 2 h, then reaerated or allowed to remain hypoxic. The newly formed DNA of hypoxic or reaerated cells was labeled with [3H]thymidine using different pulse and pulse/pulse-chase protocols. The chain length distribution of the labeled DNA molecules was analysed by sedimentation after lysing the cells on the top of alkaline sucrose gradients. The results indicated that the hypoxia effectively and reversibly suppressed the initiation of new replication units. Initiation, growth and integration of Okazaki pieces into active replicons was not noticeably affected. In marked contrast to aerobic cells, the use of hypoxic cells allows the separation of Okazaki pieces as a distinct class of pulse labeled short DNA chains. Short daughter DNA of very recently initiated replicons did not interfere at pulse times shorter than 4 min. For examination of the newly initiated replicons it seems favourable to trigger a burst of initiations by reaeration.     

Effect of aging on EGF-stimulated replication of specific genes in rat hepatocytes

EGF-stimulated replication of specific genes was examined in primary hepatocyte cultures from mature (6 months) and senescent (24 months) rats. Basal and EGF-stimulated [³H]thymidine incorporation and DNA polymerase α activities, as well as total cellular DNA, were also assessed. The genes examined were dihydrofolate reductase (DHFR) and c-myc, as well as total mitochondrial DNA (mt DNA). Although [³H]thymidine incorporation, DNA polymerase α activity, total cellular DNA, DHFR, and c-myc gene specific DNA replication stimulated by EGF are reduced with age, mt DNA replication is not affected by either EGF or age. Chromosomal DNA replication is mediated mainly by DNA polymerase α while mt DNA replication is mediated by its own DNA polymerase γ. Thus, the age-related decline in stimulated DNA replication appears to be associated mainly with the DNA polymerase α activation pathway. J. Cell. Physiol. 176:32–39, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

DNA nucleotide excision-repair synthesis is dependent of pertubations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [³H]TTP from [³H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [³H]TTP pool was formed within 30 min of the addition of [³H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [³H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [³H]TTP pool size in cells exposed to 20 M/m² and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [³H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [³H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [³H]TTP pool.     

DNA REPLICATION: DIRECTION AND RATE OF CHAIN GROWTH IN MAMMALIAN CELLS

Using pulse labeling techniques with [³H]thymidine or [³H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [³H]thymidine of high specific activity, followed by [³H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15–23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation.     

Direction of DNA replication in mammalian cells

We have re-examined the direction of DNA synthesis in mammalian cells by means of pulse-labeling with [³H]thymidine and DNA autoradiography. Our results show that, whether or not the cells are treated with 5-fluoro-deoxyuridine, and whether they are labeled first with high specific activity [³H]thymidine and then with low, or vice versa, most (? 90%) of the unambiguous autoradiographic patterns can be explained by bidirectional replication but not by unidirectional replication.We also find that in autoradiographic experiments using two different specific activities of [³H]thymidine, obvious differences in grain density are obtained only when the difference in specific activity is threefold or more. Thus, the apparently contradictory findings of Lark et al. (1971) can be explained by the low difference in specific activity used by those authors.     

Pulse-Labeling of Kinetoplast DNA: Localization of 2 Sites of Synthesis Within the Networks and Kinetics of Labeling of Closed Minicircles*

SYNOPSIS. Short pulse-labeling of log phase Crithidia fasciculata cells with [³H]thymidine allowed the autoradiographic visualization of 2 sites of replication of kinetoplast DNA situated at the periphery of the networks and separated by 180°. Longer pulse-labeling led to the previously reported total peripheral labeling pattern. Pulse-labeled networks possess an intermediate density in ethidium bromide-CsCl equilibrium gradients between the densities characteristic of closed networks and open or linear DNA. Removal of ethidium bromide by several methods and treatment of intermediate band networks with RNase and pronase had no effect on the equilibrium rebanding pattern. Closed minicircles of Leishmania tarentolae are not labeled by a short pulse of intact cells with [³H]thymidine. A chase of ～ 3–4 hr is required for the appearance of radioactivity in closed minicircles, a time delay which implies the existence of intermediate events between replication and eventual covalent closure of the minicircles.     

Synthesis and turnover of Euglena gracilis mitochondrial DNA

Replication of mitochondrial DNA was investigated by a density transfer experiment in a strain of Euglena gracilis lacking chloroplast DNA. DNA was uniformly labeled in a medium containing ³²P-labeled inorganic phosphate and [³H]adenine in the presence of the heavy-density label and transferred to a medium containing ³²P-labeled inorganic phosphate but no [³H]adenine following removal of the heavy-density label. Replication of nuclear DNA within these cells was used as an internal control. The densities and ratios of the peaks of nuclear DNA were those expected for a strict semiconservative replication. In contrast, replication of mitochondrial DNA was dispersive, as illustrated by the following results: (1) both native and denatured mitochondrial DNA exhibited a single density peak at 1.1 and 2.2 cell doublings after the density transfer. (2) The specific activity of ³H-labeled DNA varied across the peak of native or denatured DNA, indicating a heterogeneous population of molecules exhibiting different degrees of density and radioisotope labeling. This dispersive replication could involve either multiple recombination events or extensive turnover of the DNA or a mixture of both. Extensive dispersion of the sample obtained at 1.1 cell doublings after the density transfer is shown by the persistence of the same peak density for duplex DNA reduced to a molecular weight of 6 × 10⁵ by shearing.Two measures of the rate of replication of mitochondrial DNA were obtained from the densities of native duplex DNA and the rate of decrease in ³H-specific activities of duplex DNA during the experiment. The average of these rates indicates that mitochondrial DNA replicates at least 1.5 times as fast as nuclear DNA. Since there is a constant ratio of mitochondrial DNA:nuclear DNA in a logarithmic culture, mitochondrial DNA was calculated to have a half-life of 1.8 cell doublings.     

RNA primers in Simian virus 40 DNA replication. II. Distribution of 5' terminal oligoribonucleotides in nascent DNA.

A cell-free simian virus 40 (SV40) DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages were found to copurify with SV40 replicating DNA. These linkages were identified by transfer of a fraction of the ³²P from the 5′ position of a deoxyribonucleotide to 2′(3′)rNMPs upon either alkaline hydrolysis or RNAase T₂ digestion of SV40 replicating [³²P]DNA. Alkaline hydrolysis also exposed 5′ terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with P1 nuclease. The RNA-DNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5′ termini (RNA primers). The density of a portion of the Okazaki pieces in potassium iodide gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a defined length in the presence of either RNAase T₂ or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least seven-residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that one-third to one-half of them contained rN-³²P-dN linkages, the oligoribonucleotides must be covalently attached to the 5′ ends of nascent DNA chains. Although the significance of two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers.Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in nascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5′ end of growing daughter strands.³²P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII + III restriction fragments of SV40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences.