Structure of F-actin needles from extracts of sea urchin oocytes

The mouse L-cell line LD maintains its mitochondrial DNA genome in the form of a head-to-tail unicircular dimer of the monomeric 16,000 base-pair species. This situation permits a comparison of the mechanism of replication of this dimeric molecule with our previous studies of replication of monomeric mouse L-cell mitochondrial DNA. Whereas monomeric mitochondrial DNA requires about one hour for a round of replication, the dimeric molecule requires almost three hours. Denaturing agarose gel electrophoretic analyses of replicative intermediates reveals several discrete size classes of partially replicated daughter strands of dimeric mitochondrial DNA. This suggests that replication occurs with specific discontinuities in the rate of daughter strand synthesis. The strand specificity of these daughter strands was determined by hybridization with ³²P-labeled DNA representing either the heavy or light strand mitochondrial DNA sequence. The sizes and strand specificities of these discrete daughter strands indicate that the same set of control sequences is functional in both dimer and monomer mitochondrial DNA replication.Immediately following a round of replication, the majority of dimeric mitochondrial DNA molecules contain displacement loops, as assessed by their sensitivity to nicking within the displaced DNA strand by single-strand DNA specific S₁ nuclease under conditions which leave supercoiled DNA intact. This result is in contrast with the conformation of newly replicated monomeric mitochondrial DNA molecules, which lack both superhelical turns and displacement loops. This indicates that dimeric mitochondrial DNA proceeds through a different series of post-replicative processing steps than does monomeric mitochondrial DNA. We postulate that intermediates at late stages of dimeric mitochondrial DNA replication contain displacement loops which remain intact following closure of the full-length daughter strands.     

Size distribution of DNA replicative intermediates in bacteriophage P4 and in Escherichia coli.

We have tested the hypothesis that Okazaki fragment replicative intermediates have defined termini using as a model system the in vivo DNA replication of the tiny bacteriophage P4. The kinetics of formation of intermediates in P4 DNA replication have been investigated. P4 DNA replication in DNA polymerase I-deficient mutants generates Okazaki fragments with a size distribution similar to that in uninfected cells. When P4-derived Okazaki fragments are resolved by agarose gel electrophoresis, no discrete size classes appear. This finding is incompatible with sequence-specific models of Okazaki fragment formation but supports the view that these replication intermediates are initiated and terminated at random locations on the P4 chromosome.     

Intermediate in SV40 DNA Chain Growth

PREVIOUS studies of the DNA replication of simian virus 40 (SV40), an oncogenic member of the papoyavirus group, have been concerned with separation and characterization of replicative intermediates^1–4. Circular replicating intermediates have been identified for SV40^1–3, as well as for the similar replication system of polyoma viral DNA5,6. The replicative intermediates of SV40 DNA have been observed by electron microscopy to contain two forks, three branches and no free ends^1–3 as is the case for the circular replicating molecules of polyoma, bacteriophage λ⁷, Escherichia coli⁸ and colicin E1 in mini-cells^9,10. An important property of replicative intermediates of SV40 DNA that has also been observed in replicating molecules of colicin E1¹⁰ is that most molecules contain a superhelical region in the unreplicated portion of the molecule¹.     

Pol III proofreading activity prevents lesion bypass as evidenced by its molecular signature within E.coli cells

Replication of genomes that contain blocking DNA lesions entails the transient replacement of the replicative DNA polymerase (Pol) by a polymerase specialized in lesion bypass. Here, we isolate and visualize at nucleotide resolution level, replication intermediates formed during lesion bypass of a single N-2-acetylaminofluorene-guanine adduct (G-AAF) in vivo. In a wild-type strain, a ladder of replication intermediates mapping from one to four nucleotides upstream of the lesion site, can be observed. In proofreading-deficient strains (mutD5 or dnaQ49), these replication intermediates disappear, thus assigning the degradation ladder to the polymerase-associated exonuclease activity. Moreover, in mutD5, a new band corresponding to the insertion of a nucleotide opposite to the lesion site is observed, suggesting that the polymerase and exonuclease activities of native Pol III enter a futile insertion-excision cycle that prevents translesion synthesis. The bypass of the G-AAF adduct located within the NarI sequence context requires the induction of the SOS response and involves either Pol V or Pol II in an error-free or a frameshift pathway, respectively. In the frameshift mutation pathway, inactivation of the proofreading activity obviates the need for SOS induction but nonetheless necessitates a functional polB gene, suggesting that, although proofreading-deficient Pol III incorporates a nucleotide opposite G-AAF, further extension still requires Pol II. These data are corroborated using a colony-based bypass assay.     

Mechanism of mitochondrial DNA replication in mouse L-cells: kinetics of synthesis and turnover of the initiation sequence

Pulse-chase radioactive labeling experiments using thymidine kinase-plus mouse LA9 cells have shown that the 7 S mitochondrial DNA initiation sequence of mitochondrial DNA is synthesized and turned over at a faster rate than previously determined. These pulse-chase labeling experiments have also determined that the replication time of mouse LA9 cell mitochondrial DNA is one hour. The halflife of pulse-labeled 7 S mitochondrial DNA initiation sequences is approximately 70 minutes. This turnover is so rapid that at least 95% of the mitochondrial DNA initiation sequences synthesized are lost to turnover without acting as primers for expansion synthesis of the mitochondrial DNA heavy strand. The mechanism of 7 S mitochondrial DNA turnover does not lead to significant accumulation of free 7 S mitochondrial DNA single-strands within mitochondria. Resynthesis of the 7 S mitochondrial DNA initiation sequence is sufficiently rapid that the majority of mitochondrial DNA molecules are maintained as displacement loop molecules. Approximately 20% of all nucleotides polymerized into mitochondrial DNA are incorporated into the 7 S initiation sequences. The size of newly synthesized 7 S mitochondrial DNA strands varies from about 500 to 620 nucleotides. Several size classes are resolved by polyacrylamide/urea gel electrophoresis and each class has approximately the same turnover rate.Mouse LD cells maintain their mitochondrial DNA genomes as unicircular, head-to-tail dimers. Since a significant fraction of these unicircular dimers contain only one displacement loop, the size of the initiation sequence in such molecules should be twice as long if synthesis of the strand is limited by the free energy of superhelix formation. An identical array of size classes of 7 S strands is obtained from this cell line as compared to mouse LA9 cells. This indicates that the extent of 7 S mitochondrial DNA synthesis is most likely determined by a nucleotide sequence specific event.     

Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro.

Replication of bacteriophage phi 29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism. In the present paper we have used an in vitro phi 29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. Two types of replicative intermediates were observed: type I (full-length double-stranded phi 29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length phi 29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a single-stranded DNA portion spanning to the other end). Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro phi 29 DNA replication system. Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner. Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded phi 29 DNA molecules were synthesized. In addition, replication of recombinant phi 29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded phi 29 DNA molecules. These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge.     

Mode of replication of the conjugative R-plasmid RSF1040 in Escherichia coli.

Replicating deoxyribonucleic acid (DNA) molecules of plasmid RSF1040, a deletion mutant of the conjugative R plasmid R6K, appear in the electron microscope as partially supercoiled structures with two open circular branches of equal size, although open structures with three branches, two branching points and no supercoiled regions (theta structures) were also found at a lower frequency. The partially supercoiled molecules sediment more rapidly than native covalently closed circular DNA in neutral sucrose gradients and band at a position intermediate between covalently closed circular and open circular DNA in CsClethidium bromide gradients. Electron microscope measurements of the linear EcoRI-treated replicative intermediates indicate that replication can be initiated at two sites (origins) on the plasmid DNA molecule located at about 23% (alpha) and 39% (beta) of the total genome length from an EcoRI end designated arbitrarily as the "left-hand" end of the molecule. The overall replication of RSF1040 is asymmetrically bidirectional. Replication from the alpha origin proceeds first to the "right" to a unique termination site located some 55% of the total genome length from the left-hand end of the molecule. At this point replication proceeds from the alpha origin to the "left" (i.e., opposite to the original direction of replication) until replication of the molecule is completed. Replication also proceeds from the beta origin asymmetrically to the unique terminus site.     

Evidence for discontinuous replication of circular mitochondrial DNA molecules from Novikoff rat ascites hepatoma cells

Double-forked circular molecules of mitochondrial DNA (mtDNA) from rat tissues, indicated by their form and size to be replicative intermediates, are of two structurally distinct classes. Molecules of the first class are totally double stranded. Molecules of the second class are defined by one daughter segment being totally or partially single stranded. Length histograms of daughter segments measuring between 2% and 44% of the total 5-µm molecular contour were constructed from samples of both classes of replicating molecules derived from mtDNA or Novikoff rat ascites hepatoma cells. For single strand-containing molecules, the lengths fell into eight distinct, reproducible groups with mean values separated by 4.1–7.6% of the circular contour length. For totally double stranded molecules, the lengths fell into seven groups, corresponding to seven of the groups found for single strand-containing molecules. These results suggest that along at least 44% of the contour of mtDNA molecules there exist discrete points at which DNA synthesis tends to be arrested. This may indicate that there are pauses in normal mtDNA synthesis. However, as the DNA used in these experiments was isolated from mitochondrial fractions, the findings may indicate that continuation of synthesis beyond specific points on the nucleotide strands requires a factor which is not available after cell disruption.     

Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells.

Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells.     

Plasmids bearing mammalian DNA-replication origin-enriched (ors) fragments initiate semiconservative replication in a cell-free system.

Four plasmids containing monkey (CV-1) origin-enriched sequences (ors), which we have previously shown to replicate autonomously in CV-1, COS-7 and HeLa cells (Frappier and Zannis-Hadjopoulos (1987) Proc. Natl. Acad. Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low-salt cytosolic extract, poly(ethylene glycol), a solution containing the four standard deoxyribonucleoside triphosphates and an ATP regenerating system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to obtain closed circular supercoiled molecules. Replicative intermediates migrating slower than form II and topoisomers migrating between forms II and I were also included among the replication products. Replication of the ors plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase beta and gamma, and was sensitive to aphidicolin indicating that DNA polymerase alpha and/or delta was responsible for DNA synthesis. Origin mapping experiments showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be bidirectional.     

Rates of RNA chain growth in developing sea urchin embryos

The average time necessary to add a nucleotide onto growing RNA chains (step time) has been determined for several developmental stages of Strongylocentrotus purpuratus embryos. One procedure involved quantitation of isolated nucleosides derived from the 3′ end of newly synthesized chains plus total rates of nucleotide incorporation. The former values provide the number of growing chains so that rates of incorproation per RNA chain may be calculated. A second procedure involves determinations of the times required for RNA molecules of given size classes to become fully labeled. These times may be equated to the synthetic times for the particular size class [Bremer, H., and Yuan, D. (1968). RNA chain growth-rate in Escherichia coli. J. Mol. Biol.38, 163–180]. The step times for blastula and pluteus embryos is 6–9 nucleotides/sec at 15°C, which would result in an average synthetic time for heterogeneous nuclear RNA (HnRNA) molecules of about 2 × 10⁶ daltons of 12–18 min. The half-life of HnRNA in this species of sea urchins is 15–20 min, implying a transient existence for large HnRNA molecules.     

Asymmetric Okazaki piece synthesis during replication of simian virus 40 DNA in vivo.

We have pulse-labeled simian virus 40 (SV40)-infected monkey cells with ³H-thymidine (³H-dThd) and have hybridized the viral Okazaki pieces (rapidly labeled short DNA chains found during DNA replication, < 250 nucleotides long) and SV40 “intermediate sized” DNA (longer nascent strands, up to full replicon size) to the separated strands of two SV40 DNA restriction fragments, one lying to either side of the origin of bidirectional DNA replication. As much as 5 fold more Okazaki piece DNA hybridized to one strand than to the other strand of each restriction fragment. The excess Okazaki piece DNA was in the strands oriented 3′ → 5′ away from the replication origin (the strands which are expected to be synthesized discontinuously). Neither the duration of the labeling period nor the temperature of the cells during labeling significantly altered this hybridization asymmetry. With respect to the hybridization of “intermediate sized” DNA, a reverse asymmetry was detected (1.7 fold more radioactivity in the strands oriented 5′ → 3′ away from the origin for a 1 min pulse label at 22°C). The effects on these hybridization asymmetries of preincubating the infected cells with FdUrd prior to pulse-labeling were also determined.We also measured the size of the Okazaki pieces using gel electrophoresis under denaturing conditons after releasing the pieces from the filter-bound DNA strands. The size distribution of the Okazaki piece DNA from each strand was the same (～ 145 nucleotides, weight average; 200–250 nucleotides, maximum size), indicating that the hybridization asymmetry resulted from a difference in the number rather than the size of the pieces in each strand.The simplest interpretation of our results is that SV40 DNA is synthesized semidiscontinuously: the strand with 3′ → 5′ orientation away from the origin is synthesized in short Okazaki pieces which are subsequently joined together, while the strand with 5′ → 3′ orientation away from the origin is synthesized continuously. Some models of two-strand discontinuous synthesis, however, cannot be ruled out.     

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.     

Interplay of DNA supercoiling and catenation during the segregation of sister duplexes

The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo.     

Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis

We had earlier characterized the nascent DNA synthesized in permeable cells of Bacillus subtilis in the presence of 5-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP as being substituted at its 5' end with a ribonucleotide moiety of the sequence pApG(pC)1-2 DNA. In this paper, we examine the origin and turnover of the DNA-linked ribonucleotide and its relationship to DNA replication. At least 50% of the RNA-linked nascent DNA chains served as guanylate acceptors when incubated with GTP and the eukaryotic capping enzyme, indicating the presence of 5'-terminal di- or triphosphate groups and suggesting that the RNA moiety is synthesized de novo and is not a degradation product. In nascent DNA produced without limitation of chain growth by dideoxyATP, the degree of terminal ribonucleotide substitution was reduced by 50%, consistent with a linkage between RNA primer removal and DNA chain growth. Such a relationship was demonstrated directly by examining the RNA primer content of nascent DNA synthesized in the absence of dideoxyATP as a function of DNA chain length. As the DNA size increased from 40 to 200 nucleotide residues, the extent of RNA substitution declined from 80% to nearly 0%. Endgroup analysis showed that the loss of RNA was accompanied by a gradual shift from predominantly adenylate residues to 5'-terminal guanylate, consistent with a stepwise removal of ribonucleotides from the 5' end. Evidence that the nascent mercurated DNA synthesized under our experimental conditions was indeed a replicative intermediate came from the study of the time course of DNA chain growth and pulse-chase experiments. In the presence of the DNA ligase inhibitor NMN, mercurated DNA accumulated in two size classes with average length of approximately 750 and 8000 nucleotide residues, presumably representing the mature size of intermediates in discontinuous DNA synthesis. Comparison with the DNA size range at which the loss of the 5'-terminal RNA moiety occurred (40 to 200 residues) indicated that the processing of RNA primers occurred at an early stage during DNA chain elongation, and that moderate size intermediates in discontinuous DNA replication (greater than 200 nucleotides) have already lost their RNA primers.     

Kinetoplast maxicircle DNA replication in Crithidia fasciculata and Trypanosoma brucei.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5',8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of kinetoplast DNA networks revealed intact maxicircle theta structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.     

Yeast and mammalian replication intermediates migrate similarly in two-dimensional gels

In the budding yeast, Saccharomyces cerevisiae, DNA replication initiates at specific, discrete chromosomal locations. At each initiation site, a single small replication bubble is generated, which subsequently expands at Y-like replication forks. We wanted to know whether other eukaryotic organisms utilize similar initiation mechanisms. For this purpose, replication intermediates (RIs) from three different organisms (Schizosaccharomyces pombe, Chinese hamster and human) were mixed individually with RIs from S. cerevisiae and then subjected to two-dimensional (2D) gel electrophoresis under conditions known to resolve molecules having different structures. All of the RIs detected by the hybridization probes we used for each organism migrated nearly identically to specific RIs of similar size from S. cerevisiae, implying that the detected RIs from all the studied organisms have very similar structures and may therefore employ the same basic initiation mechanism.     

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures

Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 m. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4–6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4–6S) and large (20–30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm³. Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 m.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.Abbreviations BUdR 5-Bromodeoxyuridine - FUdR 5-Fluorodeoxyuridine     

Size distribution and maturation of newly replicated DNA through the S and G2 phases of physarum polycephalum

The size distribution of newly made DNA and the dynamics of size maturation of progeny DNA molecules were studied in the synchronous S and G2 phases of Physarum polycephalum. Pulse labeling of DNA and analysis of the products on alkaline sucrose gradients showed that synthesis of primary replication units (which will also be referred to as “Okazaki” fragments) occurred throughout the S period. Pulse and pulse-chase experiments revealed a distinct pattern of size maturation. An apparently linear increase in molecular weight of progeny DNA molecules during the first hour of the S phase occurred at a rate of approximately 4–5 × 10⁵ daltons per min at 26°C, corresponding to the joining of 6–8 Okazaki fragments. The resulting 35–45S (1.1–2.2 × 10⁷ daltons) DNA molecules may correspond to the Physarum “replicon.” The further size increases of the newly made DNA appear to occur in steps, possibly reflecting a clustering of isochronous replicons along the chromatide. These observations are discussed with regard to mechanisms of DNA replication and size maturation.