RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins

Linda J. Roman, Dan A. Dixon, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB). RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA. RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule. At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion. The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo.

Original languageEnglish (US)
Pages (from-to)3367-3371
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number8
StatePublished - 1991
Externally publishedYes

Fingerprint

Rec A Recombinases
Exodeoxyribonuclease V
Joints
Single-Stranded DNA
Escherichia coli
DNA
Homologous Recombination
DNA-Binding Proteins
Mutant Proteins
Phenotype
In Vitro Techniques

Keywords

  • Genetic recombination
  • Homologous pairing
  • Single-stranded DNA-binding protein

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins. / Roman, Linda J.; Dixon, Dan A.; Kowalczykowski, Stephen C.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 88, No. 8, 1991, p. 3367-3371.

Research output: Contribution to journalArticle

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N2 - We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB). RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA. RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule. At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion. The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo.

AB - We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB). RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA. RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule. At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion. The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo.

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