Site- and strand-specific nicking at oriT of plasmid R100 in a purified system: Enhancement of the nicking activity of tral (helicase I) with traY and IHF

Susumu Inamoto, Hirokazu Fukuda, Tatsuhiko Abo, Eiichi Ohtsubo

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

We developed a purified system for reproducing the nicking reaction at the site 59 base pairs upstream of the TraY protein binding site, sbyA, in the oriT region of plasmid R100. Nicking at oriT occurred efficiently in the presence of the plasmid-encoded proteins, Tral and TraY, integration host factor (IHF), and Mg2+, but inefficiently in the presence of the Tral protein and Mg2+. The products were complex DNA molecules with a protein covalent-ly linked with the 5' end of the nick in the strand, which is supposed to be transferred during conjugation. The same complex DNA molecules were formed in the presence of the Tral protein alone, indicating that the protein attached at the 5' end of the nick is the Tral protein. Stimulation of the nicking reaction by the TraY protein and by IHF, whose binding site has been mapped between the nicking site and sbyA, indicates that DNA bending is important in the formation of the complex including the Tral and TraY proteins at oriT.

Original languageEnglish
Pages (from-to)838-844
Number of pages7
JournalJournal of Biochemistry
Volume116
Issue number4
Publication statusPublished - Oct 1994
Externally publishedYes

Fingerprint

Integration Host Factors
Plasmids
Enhancement
Proteins
Protein
DNA
Binding sites
Binding Sites
Molecules
Factors
Protein Binding
Base Pairing
Conjugation

Keywords

  • Conjugation
  • Endonuclease
  • Origin of transfer
  • Sex factor

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Radiology Nuclear Medicine and imaging
  • Molecular Biology
  • Biochemistry

Cite this

Site- and strand-specific nicking at oriT of plasmid R100 in a purified system : Enhancement of the nicking activity of tral (helicase I) with traY and IHF. / Inamoto, Susumu; Fukuda, Hirokazu; Abo, Tatsuhiko; Ohtsubo, Eiichi.

In: Journal of Biochemistry, Vol. 116, No. 4, 10.1994, p. 838-844.

Research output: Contribution to journalArticle

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N2 - We developed a purified system for reproducing the nicking reaction at the site 59 base pairs upstream of the TraY protein binding site, sbyA, in the oriT region of plasmid R100. Nicking at oriT occurred efficiently in the presence of the plasmid-encoded proteins, Tral and TraY, integration host factor (IHF), and Mg2+, but inefficiently in the presence of the Tral protein and Mg2+. The products were complex DNA molecules with a protein covalent-ly linked with the 5' end of the nick in the strand, which is supposed to be transferred during conjugation. The same complex DNA molecules were formed in the presence of the Tral protein alone, indicating that the protein attached at the 5' end of the nick is the Tral protein. Stimulation of the nicking reaction by the TraY protein and by IHF, whose binding site has been mapped between the nicking site and sbyA, indicates that DNA bending is important in the formation of the complex including the Tral and TraY proteins at oriT.

AB - We developed a purified system for reproducing the nicking reaction at the site 59 base pairs upstream of the TraY protein binding site, sbyA, in the oriT region of plasmid R100. Nicking at oriT occurred efficiently in the presence of the plasmid-encoded proteins, Tral and TraY, integration host factor (IHF), and Mg2+, but inefficiently in the presence of the Tral protein and Mg2+. The products were complex DNA molecules with a protein covalent-ly linked with the 5' end of the nick in the strand, which is supposed to be transferred during conjugation. The same complex DNA molecules were formed in the presence of the Tral protein alone, indicating that the protein attached at the 5' end of the nick is the Tral protein. Stimulation of the nicking reaction by the TraY protein and by IHF, whose binding site has been mapped between the nicking site and sbyA, indicates that DNA bending is important in the formation of the complex including the Tral and TraY proteins at oriT.

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