Chromosome Replication in Toluenized Bacillus subtilis Cells

Matsushita, Tatsuo; White, Kalpana; Sueoka, Noboru

doi:10.1038/newbio232111a0

Cited by 71 publications

(23 citation statements)

References 12 publications

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“…Recent studies of toluene-treated Bacillus subtilis give results consistent with those in this report (15).…”

Section: Methodssupporting

confidence: 81%

“…1A, and may be due to Pol I (4). This light material contains many single-strand breaks, since it ranges in size from 15 X 106 to <105 daltons in alkaline sucrose. In contrast, the DNA replicated semiconservatively, either before or after toluene treatment, showed a molecular weight of 8 X 106 in alkali and 30 X 106 when native, which rules out the possibility that the hybrid density material was composed of small fragments.…”

Section: Methodsmentioning

confidence: 99%

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Toluene-Treated Escherichia coli Replicate Only That DNA Which Was About To Be Replicated In Vivo

Burger

1971

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

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DNA density-transfer experiments with Escherichia coli strain B/r indicate that the DNA synthesized from exogenous substrates after treatment with toluene is made semiconservatively from that region of the chromosome about to be replicated at the time of toluene treatment. Up to 15% of the chromosome is replicated, at a rate 10-20% of that in vivo. A minor fraction of nonconservatively replicated DNA is readily distinguished.It seems likely that DNA polymerase (1) (Pol I, EC 2.7.7.7) is involved not in the replication but in the repair of DNA in Escherichia coli (2,3). Experiments involving relatively gentle treatment of E. coli with toluene have recently revealed another polymerase activity (4). If bacterial mutants unable to synthesize DNA at an elevated temperature are treated with toluene, the new polymerase activity assayed in such toluenized cells is much reduced at the same restrictive temperature (4-6). Two further similarities between replication in vivo and in toluene-treated cells are reported below. We demonstrate the transfer of radioactive label from light chromosomal DNA to that of a hybrid density upon incubation of toluene-treated cells with a mixture of deoxynucleoside triphosphates containing bromodeoxyuridine triphosphate (BrdUTP) instead of dTTP. Only that part of the chromo-. some about to be replicated when toluene treatment begins exhibits this density transfer. MATERIALS AND METHODSThe methods of Moses and Richardson (4) dissolved in 10 mM Tris-10 mM sodium EDTA buffer (pH 8.5); concentrations were determined and adjusted by the use of a Zeiss refractometer. Growth media contained minimal salts (9) plus 2 mg/ml of glucose, 50 ,g/ml of D-histidine, and 3 ug/ml of thymine. Cells (E. coli, B/r, thy-, his-designated CW6, from C. B. Ward) were grown at 37°C with shaking, to a density of 2-3 X 108 cells/ml, then harvested by centrifugation (2000 X g) at 4°C, and resuspended at 5 X 109 cells/ml in 50 mM potassium phosphate buffer (pH 7.4) containing 10 mM MgCl2. The suspension was shaken with 1% toluene for 5 min at 37°C and then diluted 4.5-fold by the addition of a mixture yielding 13 mM MgCl2, 0.13 mM NAD, 1.3 mM ATP, 33 ,MM (each) dATP, dGTP, and BrdUTP, and 20MAM [3H]dCTP (10 ,uCi/ml), in 0.9 ml of 70 mM potassium phosphate buffer (pH 7.4). Incubation at 37°C was halted after 20 min by precipitation with C13CCOOH (4) or by chilling if density analywis was desired. The chilled cells were sedimented three times at 5000 X g from 10 mM Tris-10 mM sodium EDTA buffer (pH 8.5), then resuspended in 1 ml, with Pronase (2 mg/ml) and sodium dodecyl sulfate (0.5%) added. Lysis occurred during 10 min at 450C. After 2 hr more at 37°C, the lysates were passed 20 times through a 21-gauge hypodermic needle to shear the DNA, and clarified by centrifugation at 20,000 X g. Lysates (0.9 ml) were added to 9 ml of CsCl (density 1.80 or 1.82 g/cc) and centrifuged 50 hr at 25°C in a Beckman type 50 rotor at 34,000 or 39,000 rpm, respectively, to resolve light from hybrid DNA or each from heavy as well. Mo...

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“…Recent studies of toluene-treated Bacillus subtilis give results consistent with those in this report (15).…”

Section: Methodssupporting

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Toluene-Treated Escherichia coli Replicate Only That DNA Which Was About To Be Replicated In Vivo

Burger

1971

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

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“…The semiconservative, ATP-dependent, replicative DNA synthesis observed in toluenized bacteria resembles in vivo DNA replication in many respects (1)(2)(3)(4). The modes of DNA synthesis that occur in toluenized bacteria in the absence of ATP have received less attention.…”

mentioning

confidence: 99%

Ultraviolet-Stimulated DNA Synthesis in Toluenized Escherichia coli Deficient in DNA Polymerase I

Masker

Hanawalt

1973

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

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Ultraviolet (UV)-stimulated nonconservative DNA synthesis has been observed in E. coli rendered permeable to nucleoside triphosphates by exposure to toluene. This synthesis is detected in toluenized cells in which the background of UV-independent DNA synthesis is reduced by use of dnaB mutants temperature-sensitive for DNA replication and additionally deficient in DNA polymerase I. UV-stimulated nonconservative synthesis is also seen in polA dnaE mutants that are deficient in two of the three known DNA polymerases. The observed UVstimulated repair-like synthesis requires the presence of the four deoxyribonucleoside triphosphates and ATP. This mode of synthesis appears to differ from the ATPindependent nonconservative DNA synthesis previously reported to occur in toluenized bacteria.Recent attempts to elucidate the mechanism of DNA replication in bacteria have demonstrated both repair-like and replicative-like DNA synthesis in cells made permeable to nucleoside triphosphates and other small molecules by exposure to toluene (1). The semiconservative, ATP-dependent, replicative DNA synthesis observed in toluenized bacteria resembles in vivo DNA replication in many respects (1-4). The modes of DNA synthesis that occur in toluenized bacteria in the absence of ATP have received less attention. This repair-like synthesis is most readily observed in strains with normal levels of DNA polymerase I, and it can be enhanced by treatment with DNase during toluenization (1). Although it was suggested that this nonconservative synthesis might be similar to the repair resynthesis observed as a consequence of ultraviolet (UV) damage to DNA (1), no study of DNA synthesis in toluenized bacteria after UV irradiation has yet been reported.In spite of the apparent involvement of DNA polymerase I in the ATP-independent DNA synthesis in permeabilized cells, doubts remain as to whether this polymerase is responsible for observably large amounts of repair resynthesis in vivo. In vivo studies (5, 6) have suggested that DNA polymerase I performs only a limited amount of resynthesis in the excision-repair of UV-induced dimers, and that it produces repair patches consisting of only a few (< 30) nucleotides (5). The recA recB gene products appear to be involved in a pathway of excision-repair in which longer patches are produced (6). In view of the uncertainties surrounding the exact mechanisms of excision-repair resynthesis, the question of whether toluenized bacteria can perform repair resynthesis of UV damage appears to be significant. Moreover, such a quasi-in vitro system offers a potentially powerful tool for investigating the enzymatic basis of excision-repair. 129 We report here a study of DNA synthesis in toluenized mutants of Escherichia coli that have suffered UV damage. Our experiments have revealed that UV-stimulated DNA synthesis can be observed in toluenized E. coli if special conditions are imposed to eliminate the high background of nonconservative DNA synthesis that occurs even in unirradiated cells. The observed U...

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“…Permeabilization of bacterial cells has recently been attained with several organic solvents. For example, bacteria treated with toluene have successfully :been used in studies of deoxyribonucleic acid (DNA) replication (12,15) and repair (15) and of ribonucleic acid (RNA) synthesis (16). So far such systems have not been extensively developed for eukaryotic cells, where it has been shown that isolated cell nuclei (5,40,21) and cellular ghosts containing nuclei (20) are very useful tools for studying transcription and maturation of specific RNA species.…”

mentioning

confidence: 99%

Ribonucleic Acid Synthesis Dependent on Exogenous Triphosphates in Nystatin-Treated Cells of Kluyveromyces lactis

Badaracco

Cassani

1976

Antimicrob Agents Chemother

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Kluyveromyces lactis cells treated with nystatin became permeable to ribonucleoside triphosphates. Although not viable, nystatin-treated cells were capable of sustaining ribonucleic acid synthesis. The system depended on the presence of divalent ions and the four nucleoside triphosphates, and was strongly stimulated by ammonium sulfate. The system utilized endogenous deoxyribonucleic acid as template. Synthesis of ribonucleic acid was associated with the cells and probably occurred internally.

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Chromosome Replication in Toluenized Bacillus subtilis Cells

Cited by 71 publications

References 12 publications

Toluene-Treated Escherichia coli Replicate Only That DNA Which Was About To Be Replicated In Vivo

Toluene-Treated Escherichia coli Replicate Only That DNA Which Was About To Be Replicated In Vivo

Ultraviolet-Stimulated DNA Synthesis in Toluenized Escherichia coli Deficient in DNA Polymerase I

Ribonucleic Acid Synthesis Dependent on Exogenous Triphosphates in Nystatin-Treated Cells of Kluyveromyces lactis

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