In Vivo Mechanism of DNA Chain Growth

Okazaki, Reiji; Okazaki, Tuneko; Sakabe, K.; Sugimoto, Kazunori; Kainuma, Ritsu; Sugino, Akio; Iwatsuki, Norio

doi:10.1101/sqb.1968.033.01.017

Cited by 213 publications

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“…Our results are best interpreted in terms of the Okazaki hypothesis (20) (9). It does seem necessary to postulate some special mechanism that will yield fragments of a particular density rather than the distribution of densities between hybrid and light expected from random shearing around transition points.…”

Section: Discussionmentioning

confidence: 89%

Accumulation of an Intermediate in DNA Synthesis by HEp.2 Cells Treated with Methyl Methanesulfonate

Kato¹,

Strauss²

1974

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial DNA replication involves as intermediates structures with single-stranded regions at the growing point (1-3). Newly synthesized, pulse-labeled DNA that behaves on hydroxyapatite and/or nitrocellulose as though it contained single-stranded regions has also been observed in mammalian cells (4-8). Chromatography on benzoylated, naphthoylated DEAE-cellulose permits the separation of such single-strandcontaining intermediates in DNA synthesis that accumulate after treatment with the monofunctional alkylating agent, methyl methanesulfonate (MMS, ref. 9). We report here the isolation from HEp. 2 cells incubated with BrdU of a fraction of newly synthesized DNA whose banding characteristics in CsCl gradients and response to incubation with a singlestrand-specific nuclease indicate that it contains singlestranded regions in both parental and newly synthesized DNA. Treatment of cells with MMS at concentrations that inhibit DNA synthesis increases the proportion of pulselabeled material appearing in this fraction. We propose that the fraction collected at a density of 1.715 g/cm8 in CsCl is enriched for normal intermediates in DNA synthesis and that alkylation-induced lesions have the effect of immobilizing and accumulating a replicating structure. into BrdU-FdU medium to a concentration of 10 mM. Cells were harvested as described (11).DNA Extraction. Cells were harvested and washed in phosphate-buffered saline (11), washed in 0.15 M NaCl + 0.015 M sodium citrate, and resuspended in saline-citrate at approximately 4.0 X 106/ml. Single-stranded, newly synthesized DNA is preferentially adsorbed to the interface of organic solvent extractions (13,14). Our preparations must, therefore, be done at high cell density (15), which makes it possible to avoid the losses in recovery reported by Hanawalt and Ray (16). Sodium dodecyl sulfate was added to 0.2%, and extraction-was carried out as reported (11).CsCl Density Centrifugation. DNA (30-60 gg), sheared by passage three times through a 22-gauge needle, was centrifuged though a CsCl gradient that was collected and processed as described (11). Alkaline gradients were prepared by the dropwise addition of 0.1 M NaOH to the DNA sample to bring the pH to 12 before addition of CsCl.Exonuclease Treatment. Fractions from CsCl gradients were pooled and dialyzed against 0.13 M glycine buffer, pH 8.4. Samples were incubated in a reaction mixture of 1.5 ml with 3 units/ml of Escherichia coli exonuclease I (supplied by Dr. N. Cozzarelli) in 13 mM MgCl2 and 3.3 mM 2-mercaptoethanol for 1.5 hr at 37°. Nuclease susceptibility was measured by comparison of the radioactivity solubilized by cold and hot 5% trichloroacetic acid. This preparation of exonuclease I, although completely specific for single-stranded DNA, includes some endonuclease activity (9). At least 90% of denatured DNA was converted to acid-soluble fragments after treatment with enzyme. 1969Abbreviation: MMS, methyl methanesulfonate.

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Section: Discussionmentioning

confidence: 89%

Accumulation of an Intermediate in DNA Synthesis by HEp.2 Cells Treated with Methyl Methanesulfonate

Kato¹,

Strauss²

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…In bacteria, at least some of the newly synthesized DNA appears as short chains (Okazaki fragments) that are subsequently joined to long molecules (1)(2)(3). Whether such discontinuous replication occurs on both strands or only on one is an open question.…”

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confidence: 99%

Size Distribution and Molecular Polarity of Newly Replicated DNA in Escherichia coli

Louarn

Bird

1974

Proc. Natl. Acad. Sci. U.S.A.

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Newly synthesized DNA, in E. coli lysogenic for the phage X, was labeled by short pulses of [ In bacteria, at least some of the newly synthesized DNA appears as short chains (Okazaki fragments) that are subsequently joined to long molecules (1-3). Whether such discontinuous replication occurs on both strands or only on one is an open question. Evidence, recently reviewed by Klein and Bonhoeffer (4), has been introduced for both possibilities. In order to resolve this question, in the case of Escherichia coli replication, we have studied the molecular polarity of newly synthesized DNA at a given point on the E. coli chromosome, the prophage lambda. The passive replication (5) of this prophage by the chromosomal machinery occurs in a known direction (6), the polarity of integration is known (7,8) and the DNA strands can be easily separated (9). These properties have allowed us to study, by DNA -DNA hybridization, the size distribution and the molecular polarity of the DNA made during exposure to short pulses of tritiated thymidine.EXPERIMENTAL DESIGN Fig. 1 shows two possible models for the manner in which the new DNA strands are formed (1). In model A, (continuous-discontinuous replication) synthesis occurs continuously in the 5' -> 3' direction by addition of nucleotides, to long DNA chains, at the 3' end. Replication in the 3' --5' direction occurs by synthesis of short chains (5' --3', i.e., backward relative to the movement of the replication fork) that are subsequently joined to long DNA.Abbreviation: SSC, standard saline-citrate solution (0.15 M sodium chloride-0.015 M sodium citrate, pH 7); 6 X SSC means that the concentration of the solution used is 6 times that of the standard saline-citrate solution. In model B (discontinuous replication) both strands are synthesized first as short chains then are joined to long DNA.We have isolated the radioactive short fragments made during a short pulse of radioactive thymidine in a strain lysogenic for X, and hybridized these fragments with the separated strands of the phage DNA. Model B predicts that radioactive fragments will hybridize to both strands of phage X, whereas model A predicts that fragments will hybridize only to strand I of X and long DNA will hybridize to strand r.We have found that replication occurs according to model A in our polA + strain. In a polA -strain two size classes of fragments are formed: the smaller class, 10 S, corresponds to the fragments found in the polA + strains; the other class, 12-50 S, corresponds to the strand that is elongated continuously in polA + strains. MATERIALS AND METHODSBacterial and Bacteriophage Strains. The E. coli Ki2 strain CB 0129 (F-Thy-Leu-BI-) was used. Its replication pat-

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“…Product Following a short incubation with radioactive deoxynucleoside triphosphates a t 10 pM concentrations we observed equal distribution of the incorporated pulse label between short chains (about 10 S in alkali) and fast-sedimenting units (> 16 S). Simil a d y , Okazaki and collaborators [12] working with lysates of polA-cells as a DNA synthesizing system (at 10 pM concentrations of deoxynucleoside triphosphates) observed products of two discrete sues, one component sedimenting a t 13 S in alkali and another component sedimenting a t an average rate of 31 S. Pulse-chase experiments indicate that the 13 Scomponent is not the precursor ofthe 31 S-component. These results as ours might be related to the hypothesis that DNA synthesis is discontinuous and that this holds for one strand whereas the other is laid down as a continuous sequence [13-171.…”

Section: Discussionmentioning

confidence: 99%

DNA Synthesis in Nucleotide Permeable Escherichia coli Cells

Geider

Hoffmann‐Berling

1971

European Journal of Biochemistry

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Cells made permeable to nucleotides by treatment with ether synthesize DNA from exogenous deoxynucleoside triphosphates in a semi-conservative fashion. The reaction proceeds from sites of previous synthesis on the chromosome, and not detectably from new sites. Short chains appear as precursors of long units and have a mean sedimentation coefficient of about 10 S in alkali, like Okazaki-pieces when synthesized in the presence of 10 to 20p.M deoxynucleoside triphosphates, and of only 5 S when synthesized in the presence of 1 p.M dNTP. Under the former conditions short chains contain not more than half of the newly synthesized DNA; under the latter conditions they can contain more than half, the remainder of the newly synthesized DNA being in long units. Nascent short chains labelled after several minutes of synthesis in the presence of 5-bromodeoxyuridine 5'-triphosphate at high deoxynucleoside triphosphate concentrations show bimodal density distribution in an alkaline Cs,SO, equilibrium gradient with peaks of material corresponding to the density of heavy single strands and almost light single strands, respectively. The implications of these findings are discussed. Judged from controls, repair synthesis was not a source of error in these experiments.

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In Vivo Mechanism of DNA Chain Growth

Cited by 213 publications

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Accumulation of an Intermediate in DNA Synthesis by HEp.2 Cells Treated with Methyl Methanesulfonate

Accumulation of an Intermediate in DNA Synthesis by HEp.2 Cells Treated with Methyl Methanesulfonate

Size Distribution and Molecular Polarity of Newly Replicated DNA in Escherichia coli

DNA Synthesis in Nucleotide Permeable Escherichia coli Cells

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