In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family

H M Thorpe, Margaret Caroline MacHin Smith

Research output: Contribution to journalArticle

313 Citations (Scopus)

Abstract

The genome of the broad host range Streptomyces temperate phage, phi C31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coil, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required, Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coil and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites, The results show that directionality in phi C31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.

Original languageEnglish
Pages (from-to)5505-5510
Number of pages6
JournalPNAS
Volume95
Issue number10
Publication statusPublished - 12 May 1998

Keywords

  • GAMMA-DELTA RESOLVASE
  • STRAND EXCHANGE
  • ATTACHMENT SITE
  • RNA-POLYMERASE
  • TN3 RESOLVASE
  • PLASMID
  • LAMBDA
  • GENE
  • STREPTOMYCES
  • EXPRESSION

Cite this

In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. / Thorpe, H M ; Smith, Margaret Caroline MacHin.

In: PNAS, Vol. 95, No. 10, 12.05.1998, p. 5505-5510.

Research output: Contribution to journalArticle

Thorpe, H M ; Smith, Margaret Caroline MacHin. / In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. In: PNAS. 1998 ; Vol. 95, No. 10. pp. 5505-5510.
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AB - The genome of the broad host range Streptomyces temperate phage, phi C31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coil, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required, Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coil and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites, The results show that directionality in phi C31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.

KW - GAMMA-DELTA RESOLVASE

KW - STRAND EXCHANGE

KW - ATTACHMENT SITE

KW - RNA-POLYMERASE

KW - TN3 RESOLVASE

KW - PLASMID

KW - LAMBDA

KW - GENE

KW - STREPTOMYCES

KW - EXPRESSION

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SN - 0027-8424

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