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

Abstract

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found conditions.that increase the ,rate of'blunt-end joining by the T4 DNA ligase by several orders of magnitude (unpublished -data).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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