In vitro reconstitution of homologous recombination reactions

S. C. Kowalczykowski

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

The proteins essential to homologous recombination in E. coli have been purified and their individual activities have been identified, permitting biochemical reconstitution of steps that comprise the cellular recombination process. This review focuses on the biochemical events responsible for the initiation and homologous pairing steps of genetic recombination. The properties of an in vitro recombination reaction that requires the concerted action of recA, recBCD, and SSB proteins and that is stimulated by the recombination hotspot, Chi(χ), are described. The recBCD enzyme serves as the initiator of this reaction; its DNA helicase activity produces single-stranded DNA that is used by the recA protein to promote homologous pairing and DNA strand invasion of supercoiled (recipient) DNA. The SSB protein acts to trap the single-stranded DNA produced by recBCD enzyme and to facilitate pairing by the recA protein. The χ regulatory sequence acts in cis by attenuating the nuclease, but not the helicase, activity of recBCD enzyme. This attenuation assures the preservation of ssDNA produced by the DNA helicase activity and is responsible for the simulation in vitro and, presumably, in vivo. The attenuation of nuclease activity by χ results in the loss or functional inactivation of the recD subunit.

Original languageEnglish (US)
Pages (from-to)204-215
Number of pages12
JournalExperientia
Volume50
Issue number3
DOIs
StatePublished - Mar 1994

Keywords

  • and DNA strand exchange
  • DNA helicase
  • Genetic recombination
  • homologous pairing
  • nuclease
  • recA protein
  • recBCD enzyme
  • recombination hotspot
  • single-stranded DNA binding protein

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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