Appearance of a late stage during mammalian DNA replication when cells resume formation of 10 kb DNA replication intermediates

After the joining of human large DNA replication intermediates and before the appearance of mature chromatin DNA, there exists a distinct stage--'the post-elongation stage'. This stage reappears during recovery of DNA synthesis simultaneously with the reappearance of a large DNA replication intermediate, 10 kb DNA.     

Two discrete DNA replication intermediates are formed in melphalan-treated cells

Regulated and nonregulated phosphorylases were found to catalyze in a slow, orthophosphate dependent reaction the direct transfer of the glucosyl residue from α-D-glucosyl fluoride to an oligosaccharide primer. The enzyme catalyzed formation of the glucosyl residue requires stereospecific protonation of α-D-glucosyl fluoride by a Brønstedt acid. The results are interpreted by a mechanism whereby phosphate acts as a proton shuttle and the cofactor pyridoxal 5′-phosphate is required to promote the acid-base function of phosphate.     

DNA replication in asynchronous and synchronous ehrlich ascites cells in different conditions of growth

Ehrlich ascites tumour cells were labelled for DNA fibre autoradiography within the peritoneal cavity of a tumour-bearing mouse. The generation and the evaluation of the autoradiographic patterns is described and discussed. To study possible changes of the autoradiographic patterns during a natural S phase the labelling was performed in the mouse or in culture with asynchronous cells which were afterwards separated into synchronous subpopulations by zonal centrifugation. The subpopulations obtained were characterized by flow cytofluorometry in connection with the thymidine labelling index. We compared the DNA fibre autoradiographic patterns of several synchronous and asynchronous cell populations growing in the mouse or under different conditions in culture: The replicon size distributions of all populations examined were virtually the same. The fork movement rate was found to depend mainly on the metabolic condition of the cells. In culture it was significantly slower than in the mouse although a shortened S phase and therewith an increased DNA synthesis rate occurred. During a natural S phase it increased slightly, at most, while the DNA synthesis rate was considerably enhanced at the end of S. The changes in the rate of total DNA synthesis cannot account for the changes in the rate of chain growth. We conclude that the DNA synthesis rate is regulated almost exclusively by changing the replicon initiation frequency, while the fork movement rate is limited by the actual metabolic condition of the cells.     

Altered frequency of initiation sites of DNA replication in Werner's syndrome cells

Summary DNA replication of cultured fibroblasts of early passage derived from Werner's syndrome (adult progeria) patients and from normal subjects were compared by DNA fiber autoradiography. The frequency of replication initiation was decreased in Werner's syndrome cells derived from five patients compared with that in normal cells derived from three persons of different ages. The rate of DNA chain elongation did not differ between Werner's syndrome cells and normal cells.     

Ubiquinone 10 formation in Rhodospirillum rubrum under different culture conditions

The formation of ubiquinone 10 and bacteriochlorophyll (bchl) was determined in Rhodospirillum rubrum grown under different culture conditions. Transfer of chemotrophically grown cultures to photosynthetic conditions leads to the formation of the pigments until cells reach the stationary phase of growth. Bchl-synthesis initially exceeds quinone synthesis. On a cellular protein basis quinone levels first decrease by about a factor of two and subsequently increase by a factor of four. Bchl levels per protein increase until cells reach the stationary phase of growth. Quinone levels per bchl decrease rather rapidly and become constant in the growing culture. When cells were transferred under continuous phototrophic conditions to new culture medium, both pigments are formed concomitantly. While protein synthesis starts immediately, bchl and ubiquinone formation is slightly delayed. This causes a short time decrease in the amount of both pigments per cellular protein followed by an increase to a constant level. Ratios of ubiquinone per bchl are constant. The transfer of phototrophically grown cultures to chemotrophic conditions results in a complete inhibition of bchl formation while quinone synthesis resumes. Quinone cellular levels decrease slightly and then remain constant. Quinone values increase per bchl which is eventually diluted out of the cells.     

Discrete sizes of replication intermediates in drosophila cells

The size of DNA replication intermediates from Drosophila cells pulse-labeled with ³H-thymidine for 30–120 sec was determined by electrophoresis in formamide-polyacrylamide gels. Replication intermediates were formed in three discrete size classes, with median lengths of 61, 125 and 240 nucleotides. Replication intermediates in the 125 nucleotide size class occurred most frequently. Two of the three size classes may contain discrete species of replication intermediates about 90–400 nucleotides long. The data also suggested that some larger replication intermediates accumulate in pulse-labeled cells. We concluded that 61 nucleotide molecules give rise to 125 and 240 nucleotide molecules, which then form high molecular weight DNA. Mechanisms for forming these replication intermediates are discussed.     

SV40 DNA replication intermediates: Analysis of drugs which target mammalian DNA replication

The simian virus 40 chromosome, a model for the mammalian replicon, is a uniquely powerful system for the study of drugs and treatments which target enzymes of the mammalian replication apparatus. High resolution gel electrophoretic analysis of normal and aberrant viral replication intermediates can be used effectively to understand the molecular events of replication failure. These events include breakage of replication forks, aberrant topoisomerase action, failure to separate daughter chromosomes, protein-DNA crosslinking, single and double strand DNA breakage, alterations in topology and inactivation of replication intermediates. The SV40 replication system can also be used to study the recombinational events which often follow drug-induced replication failure.     

10 kb DNA replication intermediates are formed from Okazaki-fragments in human malignant melanoma cells

Human melanoma cells were synchronized with hydroxyurea. The DNA replication intermediates formed either during the hydroxyurea-treatment or after the release of the hydroxyurea-block were investigated. The intermediates were released from the parental DNA by lysing the cells in dilute alkali and were then analyzed by agarose gel electrophoresis. After the release of the hydroxyurea block radioactivity was first detected in Okazaki-fragments and then later also in larger replication intermediates (ranging in size up to 10 kb). The results indicate that the 10 kb DNA replication intermediate is derived from the Okazaki-fragments. Thus during its maturation to high molecular weight DNA the Okazaki-fragments form a large, well defined DNA replication intermediate, which later forms the high molecular weight DNA. However, it is not possible to determine whether all or only a part of the Okazaki-fragments give rise to the 10 kb DNA.     

Semiconservative DNA replication in vitro. II. Replicative intermediates of mouse P-815 cells

DNA chain growing during semiconservative replication was studied using both in vitro systems described in the preceding paper (preceding paper, ref 1) 3H-Labeled, 4-S Okazaki fragments synthesized in vivo just prior to permeabilization or lysis with Brij-58 were metabolically stable and quantitatively chased into high molecular weight DNA (20--100 S) during a subsequent incubation in vitro. Thus, DNA replication continued in vitro at the same growing points that were active in vivo. After a 20-s pulse at 30 degress C in vitro, more than 50% of incorporated radioactivity was found in the 4 S region of alkaline sucrose gradients suggesting a totally discontinuous mode of DNA chain growth. If the pulse were followed by a 1-min chase, 4-S molecules were converted into 6--12-S intermediates which upon continued incubation were joined with growing 20--100-S molecules (replicon-sized chains). Formation of all three classes of replicative intermediates, Okazaki fragments, 6--12-S intermediates, and 20--100-S molecules, occurred in vitro at least during the first 20 min. During this time, average rates of DNA chain growth and overall DNA synthesis were reduced to about the same extent, if compared to rates of intact cells. Thus, reduced chain growth rates appear to reflect primary deficiences of our in vitro systems, while initiation of replicative intermediates still occurs.     

Rapidly labeled intermediates in DNA replication in higher plants.

In detached Vicia embryos, the incorporation of [3-H] thymidine into DNA starts at about 25 h after the beginning of imbibition and reaches maximum at about 33 h. The DNA synthesized during the first replication phase was extracted. Alkaline sucrose density-gradient analyses of the DNA indicated the occurrence of several short pieces of rapidly labeled DNA having sedimentation values of approx. 10 S and 14 S, after a pulse for 5 to 10 min. Prolonged labeling and chase incubation led to a shift of the shorter fragments to longer ones of 19 S and 22 S or more, thus indicating the nature of intermediates during DNA replication of these short fragments.     

DNA replication in mammalian cells. Altered patterns of initiation during inhibition of protein synthesis

The effects of inhibition of protein synthesis by the antibiotics cycloheximide and puromycin on the initiation of DNA replication in mouse L cells were studied. Cellular DNA was pulse labeled with [3H]thymidine of high, then of low specific activity and prepared for fiber autoradiography. Autoradiograms containing multiple (up to four) replication units were analyzed. In control cells, the proportion of replication units that initiated during a 10-min, high specific activity pulse was approximately equal to the proportion initiating immediately before the pulse. The addition of cycloheximide or puromycin at the start of the pulse inhibited the frequency of initiation in that there was a decrease by up to one-third of units initiating during the pulse relative to controls. Replication direction was also altered. Addition of the antibiotics 2 h before the pulse reduced the proportion of bidirectional units observed from 0.98 to 0.70. Antibiotic treatment for 2 h also decreased initiation synchrony in that the proportion of multiunit autoradiograms on which neighboring units showed similar replication patterns (indicating temporally coordinated initiation) was reduced by one-half. These observations indicate that inhibition of protein synthesis alters the normal pattern of DNA initiation.     

Regulation of DNA replication by ATR: signaling in response to DNA intermediates

The nuclear protein kinase ATR controls S-phase progression in response to DNA damage and replication fork stalling, including damage caused by ultraviolet irradiation, hyperoxia, and replication inhibitors like aphidicolin and hydroxyurea. ATR activation and substrate specificity require the presence of adapter and mediator molecules, ultimately resulting in the downstream inhibition of the S-phase kinases that function to initiate DNA replication at origins of replication. The data reviewed strongly support the hypothesis that ATR is activated in response to persistent RPA-bound single-stranded DNA, a common intermediate of unstressed and damaged DNA replication and metabolism.     

PriA mediates DNA replication pathway choice at recombination intermediates

We report the reconstitution of the initial steps of the double-strand break-repair pathway where joint molecule formation between a duplex DNA fragment and a circular template by the combined action of RecA, RecBCD, and the single-stranded DNA binding protein provides the substrate for replication fork formation by the restart primosome and the DNA polymerase III holoenzyme. We show that PriA dictates the pathway of replication from the recombination intermediate by inhibiting a nonspecific, strand displacement DNA synthesis reaction and favoring the formation of a bona fide replication fork. Furthermore, we find that RecO and RecR significantly stimulate this recombination-directed DNA replication reaction, and that this stimulation is modulated by the presence of RecF, suggesting that the latter protein may also act as a regulator of the pathway of resolution of the recombination intermediate.     

Single-stranded replication intermediates of ribosomal DNA replicons of pea.

Replication of ribosomal DNA replicons in cells of Pisum sativum (cv. Alaska) occurs bidirectionally by displacement loops. Replication is initiated on opposite parental strands and nascent chains are elongated moving 5'----3' along each parental template. Replicative intermediates were analyzed by 2-dimensional agarose gel electrophoresis under neutral--neutral and neutral--alkaline conditions. Southern blots of ribosomal DNA fragments separated in the second dimension under neutral conditions show slowly migrating replicative fragments that hybridize with specific probes in a manner consistent with bidirectional replication. The replicative fragments are present in root meristems with cells in S phase; they are absent or few in number in meristems with cells in G2 phase. The following observations indicate that the replicative fragments are single stranded. The apparent length of the replicative fragments is not the same when separated under neutral and alkaline conditions. They contain rDNA without breaks and they do not exhibit the smaller nascent chains expected from replication bubbles and forks. They are not cleaved by restriction enzymes that require duplex DNA as substrate and they are digestible by S1 nuclease.     

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.