Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1985 May 10;13(9):3261–3271. doi: 10.1093/nar/13.9.3261

Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol.

K Hayashi, M Nakazawa, Y Ishizaki, A Obayashi
PMCID: PMC341233  PMID: 2987879

Abstract

Monovalent cations such as Na+ and K+ inhibit the activity of T4 DNA ligase. However, the extent of inhibition varies with the terminal sequence of the duplex DNA used as substrate; in many cases, ligation of DNA is completely inhibited at 200 mM. The activity of the ligase is stimulated by raising the concentration of polyethylene glycol 6000 from 0 to 15% (w/v) when NaC1 and KC1 were both absent. Ligation was reduced as the concentration of NaC1 or KC1 was raised in a mixture containing 5 or 15% PEG 6000. With 10% PEG 6000, both cohesive- and blunt-end ligation of this ligase increased at high concentrations of salt (150-200 mM NaC1, or 200-250 mM KC1). Further, with 10% PEG 6000, inter- and intramolecular ligation occurred at low salt concentrations (0-100 mM NaC1, or 0-150 mM KC1); only linear oligomers were formed by intermolecular ligation at the high concentrations.

Full text

PDF
3261

Images in this article

3263Image
on p.3263
3264Image
on p.3264
3266Image
on p.3266
3267Image
on p.3267
3268Image
on p.3268

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Fuller R. S., Kaguni J. M., Kornberg A. Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7370–7374. doi: 10.1073/pnas.78.12.7370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Laurent T. C., Preston B. N., Carlsson B. Conformational transitions of polynucleotides in polymer media. Eur J Biochem. 1974 Apr 1;43(2):231–235. doi: 10.1111/j.1432-1033.1974.tb03404.x. [DOI] [PubMed] [Google Scholar]
  3. Murray N. E., Bruce S. A., Murray K. Molecular cloning of the DNA ligase gene from bacteriophage T4. II. Amplification and preparation of the gene product. J Mol Biol. 1979 Aug 15;132(3):493–505. doi: 10.1016/0022-2836(79)90271-7. [DOI] [PubMed] [Google Scholar]
  4. Raae A. J., Kleppe R. K., Kleppe K. Kinetics and effect of salts and polyamines on T4 polynucleotide ligase. Eur J Biochem. 1975 Dec 15;60(2):437–443. doi: 10.1111/j.1432-1033.1975.tb21021.x. [DOI] [PubMed] [Google Scholar]
  5. Sgaramella V., Van de Sande J. H., Khorana H. G. Studies on polynucleotides, C. A novel joining reaction catalyzed by the T4-polynucleotide ligase. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1468–1475. doi: 10.1073/pnas.67.3.1468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Tsurimoto T., Matsubara K. Replication of lambda dv plasmid in vitro promoted by purified lambda O and P proteins. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7639–7643. doi: 10.1073/pnas.79.24.7639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Weiss B., Jacquemin-Sablon A., Live T. R., Fareed G. C., Richardson C. C. Enzymatic breakage and joining of deoxyribonucleic acid. VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4. J Biol Chem. 1968 Sep 10;243(17):4543–4555. [PubMed] [Google Scholar]
  8. Weiss B., Richardson C. C. Enzymatic breakage and joining of deoxyribonucleic acid, I. Repair of single-strand breaks in DNA by an enzyme system from Escherichia coli infected with T4 bacteriophage. Proc Natl Acad Sci U S A. 1967 Apr;57(4):1021–1028. doi: 10.1073/pnas.57.4.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Weiss B., Thompson A., Richardson C. C. Ezymatic breakage and joining of deoxyribonucleic acid. VII. Properties of the enzyme-adenylate intermediate in the polynucleotide ligase reaction. J Biol Chem. 1968 Sep 10;243(17):4556–4563. [PubMed] [Google Scholar]
  10. Wilson G. G., Murray N. E. Molecular cloning of the DNA ligase gene from bacteriophage T4. I. Characterisation of the recombinants. J Mol Biol. 1979 Aug 15;132(3):471–491. doi: 10.1016/0022-2836(79)90270-5. [DOI] [PubMed] [Google Scholar]
  11. Wold M. S., Mallory J. B., Roberts J. D., LeBowitz J. H., McMacken R. Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6176–6180. doi: 10.1073/pnas.79.20.6176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Zimmerman S. B., Pheiffer B. H. Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5852–5856. doi: 10.1073/pnas.80.19.5852. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES