Replication of colicinogenic factor E 1 DNA: evidence for a discontinuous replication mechanism

The mechanism of Col E 1 DNA replication was investigated in a plasmolysed cell system prepared from chloramphenicoltreated E. coli JC 411 (Col E 1). After pulse-labelling with (3)H-dTTP a considerable fraction of the newly synthesized DNA was recovered as single-stranded fragments. Upon alkali denaturation the pulse label was found in DNA chains sedimenting slower than unit length Col E 1 strands with a prominent peak at 5 S. During a chase with unlabeled precursors the label is transferred nearly completely into supercoiled Col E 1 DNA. DNA ligase appears to be required for the joining of the 5 S pieces since in the absence of NAD an accumulation of short fragments is observed.     

Covalent linkage between RNA and nascent DNA in the slime mold, Physarum polycephalum.

When alpha--32 P-labeled deoxyribonucleoside triphosphates are injected into plasmodia of the eukaryotic slime mold, Physarum polycephalum, they are incorporated initially into strands of DNA which are mostly less than 300 nucleotides long. Sixty minutes after injection incorporated deoxyribonucleoside triphosphates are found in much longer strands. If the short strands found two minutes after injection are denatured and centrifuged to equilibrium in a Cs(2)SO(4) density gradient, they migrate to a density slightly greater than that of single-stranded Physarum DNA. When these short strands are treated with alkali to hydrolyze RNA, a small fraction of the incorporated -32P is made acid-soluble and is identified as a mixture of the four ribonucleoside 2',3'-monophosphates. Such transfer of -32P to ribonucleotides occurs when any of the 4 alpha--32P-labeled deoxyribonucleoside triphosphates is used for injection, but the transfer is greatest with [alpha--32P]dGTP. We conclude that very short stretches of RNA are found linked through phosphodiester bonds to nascent DNA chains in Physarum polycephalum and that any of the 16 possible combinations of ribo- and deoxyribonucleotides can occur at the RNA-DNA junction.     

DNA replication in Escherichia coli: evidence for two classes of small deoxyribonucleotide chains

We have used [³H]thymidine to pulse label cultures growing at 20 or 37 °C. At these temperatures, thymidine can be used interchangeably with thymine for labeling periods of 0.1 minute or longer. Incorporation does not stop immediately when cultures are poured on to ice-cold medium-KCN mixtures, but can be stopped by pouring on to the same mixture plus pyridine.We have extracted DNA from cells pulse-labeled with [³H]thymidine and measured the number of short deoxynucleotide chains by treating them with bacterial alkaline phosphatase and labeling their 5′ ends with ³²P using [γ-³²P]ATP and polynucleotide kinase. There are at least 40 deoxynucleotide chains per bacterium (20 per replicating chromosome) whose sedimentation coefficient is less than 15 S.The small deoxynucleotide chains labeled by a pulse of [³H]thymidine behave as intermediates in the replication of DNA. They accumulate if ligase is inhibited and they disappear if their synthesis is blocked by inactivating a gene product responsible for their synthesis. In contrast, the ³²P-labeled pieces do not accumulate rapidly or disappear under the same experimental conditions. The data indicate that many of the ³²P-labeled short chains are not located at the replication fork and that they do not behave as intermediates in the replication of DNA.The size distribution of³H pulse-labeled pieces and of the short chains labeled with ³²P are apparently the same when measured by sedimentation through alkaline sucrose. About 25% of the molecules are extremely short. The remainder are distributed in such a way that, roughly, the number of pieces greater than any particular length decreases exponentially as that length increases.     

Replication of the DNA of chick embryo lethal orphan virus

Replication of the DNA of chick embryo lethal orphan virus was semi-conservative. In CsCl density gradients a portion of pulse-labelled intracellular viral DNA was more dense than mature DNA and sometimes approached the density of denatured DNA. Chromatography on benzoylated naphthoylated DEAE-cellulose also suggested that replicating viral DNA had extensive single-stranded regions. In neutral sucrose, some pulse-labelled viral DNA sedimented faster than mature DNA. Short pulses of [³H]thymidine were incorporated into fragments that sedimented at about 12 s in alkaline sucrose. As the pulse length was increased, label was found in material that sedimented faster than 12 s fragments but more slowly than the strands of mature viral DNA, and finally in full length viral DNA strands. During a “chase” in unlabelled medium, pulse-labelled intracellular viral DNA was converted to a form with properties like those of mature DNA. No closed circular structures could be detected when pulse-labelled DNA was centrifuged in CsCl in the presence of ethidium bromide. Thus the replication of this DNA, which is linear and lacks terminal repetitions detectable by exonuclease digestion and annealing, does not involve circles or concatemers in which one or both strands are continuous. However, the 5′ ends of the daughter strands cannot be completed unless the nascent DNA forms a maturation intermediate, the most likely form of which is a concatemer with staggered nicks in both strands at one genome intervals. This implies an unusual structure of the ends of the DNA, or the existence of a protein that interacts with the ends.     

The isolation and properties of a DNA-unwinding protein from Ustilago maydis.

The direction of DNA chain growth in thymine-depleted bacteria was determined by comparing the rate of release of radioactive label by Escherichia coli exonuclease I from pulse-labeled DNA chains to that of uniformly labeled DNA of the same size. Radioactive label was found to be distributed throughout the length of the pulse-labeled DNA, indicating that longer chains arise through the joining of many extremely small polynucleotide chains.     

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.     

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.     

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.     

In vitro polyoma DNA synthesis: discontinuous chain growth

Using an in vitro system for polyoma DNA synthesis from polyoma-infected mouse BALB/3T3 cells, we have shown that short pulses of radioactively labeled deoxynucleoside triphosphates are incorporated into viral replicative intermediates. Upon denaturation, the pulse-labeled replicative intermediates yield two size classes of growing DNA chains, namely a heterogeneous long class with S values up to unit viral DNA length (16 S) and a rather discrete short class of 5 S pieces. We have shown that these short fragments are involved as precursors in viral DNA chain elongation and that they can be chased into mature viral DNA. The fragments are found in replicative intermediates at all stages of replication and are therefore presumably not involved in specialized initiation or termination processes. Kinetic analysis of the appearance of radioactivity in short and long chains shows that initially approximately equal amounts are incorporated at a linear rate into the two classes. Subsequently, the rate of incorporation into long chains approximately doubles, while the amount of radioactivity in short chains reaches a plateau. This not only suggests that short chains are precursors to long chains, but that the synthesis of long chains occurs as a separate event and is not simply a result of joining of short fragments. Under the in vitro labeling conditions the time taken for radioactivity in short chains to reach a constant level can be used as a measure of the average lifetime of a 5 S piece. Our analysis indicates that there may be a considerable lag between the completion of a 5 S piece and its joining into longer DNA. Estimates of the self-annealing of the short chains showed approximately 20% self-complementarity. Thus, polyoma synthesis in vitro appears to proceed predominantly by a semi-discontinuous mechanism in which the nascent DNA on one side of the growing fork is elongated continuously, while on the other side of the fork DNA is synthesized discontinuously as 5 S fragments, which are subsequently joined. Both the short and the long chains are synthesized in the 5′ to 3′ direction.A fraction of the pulse-labeled material is found as free 3 to 5 S single-stranded DNA. These pieces are of both viral and cellular origin. A majority of them appear to be involved as precursors in DNA chain elongation.     

Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase.

A cell extract prepared from the lig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin E1 plasmid at 25 °C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from the thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25 °C to 32 °C, whereas that in the lig⁺ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the lig-ts extract, even at 25 °C.The DNA pulse-labelled with the lig-ts extract for 30 seconds at 32 °C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular molecules upon chasing with a lig⁺ extract. DNA-DNA hybridization experiments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5′-to-3′ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component.The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plasmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compound does not interfere with elongation of progeny strands. Chain elongation in the lig-ts extract at 25 °C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the lig-ts extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin E1 plasmid DNA replicate discontinuously.     

Synthesis of high molecular weight DNA strands during S phase

The organization of the mammalian S phase was studied in synchronized mouse embryo cells in terms of the spatial relationship between replication units whose synthesis is initiated at different times in S phase and the rate of assimilation of replication units into high molecular weight DNA strands.The formation of high molecular weight nascent DNA strands several replication units in length was analyzed by velocity sedimentation in alkaline sucrose gradients and by isopycnic centrifugation in alkaline Cs₂SO₄/CsCl gradients. Differential labeling with an isotopic and a density label shows that replication units synthesized at different stages of the S phase are not found within the same high molecular weight polynucleotide strand. It is thus concluded that replication units duplicated at different stages of the S phase are spatially organized in clusters along the mammalian genome.The rate of formation of high molecular weight nascent DNA strands is at least 4 to 8 times slower than that predicted from the spatial organization of replication units and the rate of chain growth within replication units. It is concluded that the process of joining of the completed nascent strands of adjacent replication units plays a major role in the rate of completion of high molecular weight strands.     

Intermediate in adenovirus type 2 replication.

Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with S1 endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 10-6 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min.     

In vitro HeLa cell DNA synthesis similarity to in vivo replication

An in vitro DNA synthesizing system, consisting of a HeLa cell lysate which incorporated dNTPs into an acid-insoluble, DNase-labile product, was optimized for incorporation per nucleus. Synthesis depended on the presence of all four dNTPs and was linear for about 15 minutes, then slowed and finally stopped after one to two hours at 37 °C. The DNA synthesized in vitro was found to be preferentially attached by covalent linkage to sites which had just been replicated in vivo. DNA fiber autoradiography of DNA labeled in vitro suggests that synthesis occurs by the replicon mechanism proposed for in vivo replication, but at a fork movement rate 50 to 60% of that in vivo.When analyzed on alkaline sucrose gradients, dNTPs appeared to be incorporated by a semidiscontinuous mechanism, with label after brief pulses (10 to 20 s) distributed about equally between a peak of Okazaki fragments and a very heterogeneous distribution of longer DNA strands. Okazaki fragments, which can be initiated in vitro, sedimented in a broad peak averaging 180 nucleotides in length.     

The Presence of Short DNA Chains in φX174 RF II in the Late Phase of Infection

In the late phase of φX174 infection, DNA was pulse-labeled and the incorporated label in RF II was chased. In resting RF II as well as active RF II there existed short DNA chains smaller than one-unit length of a linear φX174 DNA. The molecular sizes varied from 5 to 12 s as sedimented through sucrose gradients in alkaline conditions. Possible mechanisms of synthesis of φX174 progeny DNA are discussed.     

In vitro polyoma DNA synthesis: inhibition by 1-beta-d-arabinofuranosyl CTP.

The effects of 1-beta-D-arabinofuranosyl CTP (ara-CTP) on DNA replication were studied in an in vitro system from polyoma-infected BALB/3T3 cells. Ara-CTP concentrations of larger than or equal to 150 muM were found to block in vitro DNA synthesis completely, and concentrations of smaller than or equal to 0.3 muM had no inhibitory effect. Intermediate concentrations resulted in a concentration-dependent reduction of the in vitro synthesis rate. Long-term labeling with [alpha-32-P]ara-CTP demonstrated the incorporation of the analogue into cellular and viral DNA concomitantly with [3-H]TTP. In pulse-labeling experiments, at noninhibitory concentrations of the analogue, ara-CTP was incorporated into short DNA fragments and long growing strands to relatively the same extent as TTP. Partial venom phosphodiesterase digestion liberated the incoporated are-CTP at essentially the same rate as incorporated TTP, excluding a predominantly terminal incorporation, and after total venom phosphodiesterase digestion greater than 80% of the incorporated ara-CTP was recovered as 5'-ara-CMP. Analysis of the long-term in vitro viral DNA product made in the presence of partially inhibiting ara-CTP concentrations demonstrated that none of the steps leading to mature viral DNA were totally inhibited at the ara-CTP concentrations used. Pulse labeling of replicating viral DNA in the presence of ara-CTP revealed two consistent differences in the pattern found in control pulses: (i) predominant labeling of short chains (5S) with reduced amounts of radioactivity in the longer growing viral DNA strands (smaller than or equal to 16S), and (ii) a one-third to one-half reduction in size for short DNA chains labeled in the presence of ara-CTP. Release of the ara-CTP inhibition with excess dCTP resulted in covalent extension of these smaller short chans to approximately the size of regular short chains labeled in the absence of the inhibitor. Isolated short chains synthesized in the presence of ara-CTP exhibited a slightly lower degree of self-complementarity than regular short chains. The predominant labeling of short chains during pulses is, therefore, not a consequence of discontinuous growth on both sides of the replication fork. Similar results were obtained with ara-ATP and N-ethylmaleimide. The experiments indicate that ara-CTP acts primarily on DNA-polymerizing activities, affecting different DNA polymerases to varying degrees. The results are discussed in terms of the possible number and identity of polymerases involved in viral (and cellular) DNA replication.     

Unintegrated viral DNA is synthesized in the cytoplasm of avian sarcoma virus-transformed duck cells by viral DNA polymerase.

We have examined the location, structure, and mechanism of synthesis of unintegrated viral DNA present in fully transformed cultures of avian sarcoma virus-infected duck cells. De novo synthesis of the unintegrated forms several weeks after the initial infection was documented by labeling unintegrated DNA in both strands with 5-bromodeoxyuridine. The unintegrated DNA is synthesized in, and probably confined to, the cytoplasm, and it consists of duplexes of short "plus" strands (ca. 0.5 X 10(6) to 1.0 X 10(6) daltons) and "minus" strands the length of a subunit of the viral genome (ca. 2.5 X 10(6) to 3.0 X 10(6) daltons). The structure of the duplex and the mode of incorporation of density label support the hypothesis that the unintegrated DNA is synthesized from an RNA templated by virus-coded DNA polymerase.     

The periodic loss of metabolically unstable DNA during replication of chromosomal DNA of CHO cells

The fate of ³H-thymidine incorporated into newly synthesized DNA of CHO cells was analyzed by either the estimation of the incorporated radioactivity per cell or sedimentation in alkaline sucrose gradient. Under conditions in which DNA synthesis proceeded continuously, of incorporated radioactivity was periodically lost and regained during a 90 min chase, corresponding to a cyclic change in the sedimentation profiles. When DNA synthesis was inhibited by hydroxyurea no cyclic change of the incorporated radioactivity was observed. The cyclic changes were regarded as the result of an actual metabolic change in³H-labelled DNA probaly joining to one of the newly formed sister strands of DNA and the loss of radioactivity seems to require active continued DNA synthesis.     

LOCALIZATION OF MACROMOLECULES IN ESCHERICHIA COLI : II. RNA and its Site of Synthesis

The distribution of RNA in cells of E. coli 15 T^-U^- labeled with uridine-H³ was studied by methods involving the analysis of radioautographic grain counts over random thin cross-sections and serial sections of the cells. The results were correlated with electron microscope morphological data. Fractionation and enzyme digestion studies showed that a large proportion of the label was found in RNA uracil and cytosine, the rest being incorporated as DNA cytosine. In fully labeled cells the distribution of label was found to be uniform throughout the cell. The situation remained unchanged when labeled cells were subsequently treated with chloramphenicol. When short pulses of label were employed a localization of a large proportion of the radioactivity became apparent. The nuclear region was identified as the site of concentration. Similar results were obtained when cells were exposed to much longer pulses of uridine-H³ in the presence of chloramphenicol. If cells were subjected to a short pulse of cytidine-H³, then allowed to grow for a while in unlabeled medium, the label, originally concentrated to some extent in the nuclear region, was found dispersed throughout the cell. The simplest hypothesis which accounts for these results is that a large fraction of the cell RNA is synthesized in a region in or near the nucleus and subsequently transferred to the cytoplasm.