Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures

Dugaiczyk, Achilles; Boyer, Herbert W.; Goodman, Howard M.

doi:10.1016/0022-2836(75)90189-8

Cited by 391 publications

(114 citation statements)

References 18 publications

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“…The dependence upon high concentrations of background molecules and the lack of specificity with regard to these molecules is consistent with a volume-exclusion mechanism (6, 7). In addition, statistical arguments (30,31) suggest that the predominance of linear over circular DNA products when ligation occurs in high concentrations of background molecules is due to a high effective concentration of DNA termini. The reasoning is that the effective concentration of ends for intermolecular joining increases with DNA concentration, but the concentration for intramolecular cyclization is independent of DNA concentration.…”

Section: Resultsmentioning

confidence: 99%

Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli.

Zimmerman¹,

Pheiffer²

1983

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

129

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In the presence of high concentrations of any of several types-of macromolecules, DNA ligase preparations.from rat liver nuclei or from Escherichia coli actively-catalyze the bluntend ligation of DNA. This is in codtmst to the lack of activity on such substrates by these enzymes under conventional assay conditions. In addition, the previously established activity of T4 DNA ligase on blunt-ended molecules is greatly increased in the presence of high concentrations. of macromolecules. Because-such 'crowded solutions may well be: a more adequate model.for intracellular conditions than assays in dilute solutions, we. suggest that blunt-end ligation may be a widely, occurring reaction in vivo. Wlll DNA ligases that seal -single-stranded breaks in duplex DNA are found in all of the major groups of living organisms (1, 2). However, only the DNA ligase induced in Escherichia coli upon infection with T4 bacteriophage has been, demonstrated to catalyze blunt-end ligation of DNA duplexes-i.e., ligation between two fully base-paired termini of DNA duplexes ( Fig. 1) (1, 3-5). We have recently found conditions.that increase the ,rate of'blunt-end joining by the T4 DNA ligase by several orders of magnitude (unpublished -data). As will be described elsewhere, these conditions utilize high concentrations of any of a variety-of macromolecules, such as plasma.albumin, polyethylene glycol'(PEG), or Ficoll, all of which appear to be acting as volume excluders (6, 7). Under such conditions, the DNA ligases-from rat liver nuclei and from E. coli have been found -to catalyze the blunt-end ligation of DNA very actively, whereas no detectable blunt-end ligation was found in the absence of the volume excluders. Because such crowded solutions may well be a more adequate model for intracellular conditions than assays in dilute solutions (8), we suggest that blunt-end ligation may be a-widely occurring reaction in vivo.MATERIALS AND METHODS Enzymes. A exonuclease was purchased from New England BioLabs. Pvu II restriction endonuclease was a product of either New England.B-ioLabs or Bethesda Research Laboratories and T4 DNA ligase and T4,RNA ligase were from Bethesda Research Laboratories. E. coli DNA ligase fraction VII (9) was-a gift from M. Gellert. Rat liver DNA ligase fraction'IV was purified as described (10).DNA and .DNA Digestions. pBR322 DNA was either a gift from M. Gellert-or the product of Bethesda Research Laboratories. A DNA was from New England BioLabs, whereas a HindIII digest of A DNA-was obtained from Bethesda Research 'Laboratories. Digestions of pRR322 DNA with Pvu II were done under the conditions suggested by the suppliers; the digested The publication costs of this article were-defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. DNA was isolated by phenol/chloroform extraction and ethanol precipitation. E.-coliDNA (P-L Biochemicals).was labeled with 32P at-'the 551-phosphate termini of.nicks introd...

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Section: Resultsmentioning

confidence: 99%

Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli.

Zimmerman¹,

Pheiffer²

1983

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

129

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“…The relative amounts of the products by inter-and intramolecular ligation is calculated with two parameters, i and 1; i is the total concentration of DNA ends, and i is the effective concentration of one end of a DNA molecule in relation to the other end (1). The optimum condition to form linear oligomers is j/i< 1; the concentration of the DNA must be high.…”

Section: Introductionmentioning

confidence: 99%

Regulation of inter- and intramolecular ligation with T4 DNA ligase in the presence of polyethylene glycol

et al. 1986

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Polyethlene glycol (PEG) stimulates ligation with T4 DNA ligase.In 10% (w/v) PEG 6000 solutions, only intermolecular ligation is enhanced by monovalent cations, while both interand intramolecular ligation occur without their presence. Similar stimulation was also caused by divalent cations or polyamines in the PEG 6000 solutions.Such properties of the ligase could be applied to control the extent of inter-and intramolecular ligation. Ligation with cations or polyamines in 10% PEG 6000 solutions was effective for intermolecular ligation. Ligation without cations or polyamines in 6.0% to 10% PEG 6000 solutions was effective for intramolecular ligation. INTRODUCTIONThe relative amounts of the products by inter-and intramolecular ligation is calculated with two parameters, i and 1; i is the total concentration of DNA ends, and i is the effective concentration of one end of a DNA molecule in relation to the other end (1). The optimum condition to form linear oligomers

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“…Specific activity varied from 30,000 dpm/1g to 240,000 dpm/pg in various preparations. Relaxed SV40 DNA was prepared from supercoiled DNA by the method of Champoux & Dulbecco (9) The various forms of SV40 DNA were quantitated and monitored using agarose gel electrophoresis (10,11). Typical conversions were 80% for formation of Msp-nicked DNA and 98% for formation of relaxed DNA.…”

Section: Methodsmentioning

confidence: 99%

The interaction of RNA polymerase II with non-promoter DNA sites

Chandler¹,

Gralla²

1981

Nucl Acids Res

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Various complexes formed between purified RNA polymerase II and simian virus 40 DNA have been characterized with respect to rates of formation, rates of dissociation, and initial velocity of RNA synthesis. Two different types of complexes can form on intact DNA templates. One of these is formed rapidly, but is quite labile; the other forms more slowly, but is moderately stable once formed. The introduction of a single strand break into DNA leads to rapid and stable complex formation, and thus is expected to create the favored binding site.The observed properties of these complexes provide a general framework for describing the interactions of RNA polymerase II at non-promoter DNA sites. This framework appears to be similar to that established for Escherichia coli RNA polymerase interactions, suggesting that the fundamental mode of non-promoter DNA binding is similar for the bacterial, plant, and mammalian enzymes.

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Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures

Cited by 391 publications

References 18 publications

Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli.

Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli.

Regulation of inter- and intramolecular ligation with T4 DNA ligase in the presence of polyethylene glycol

The interaction of RNA polymerase II with non-promoter DNA sites

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