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

AUTOR(ES)

Roman, L J

RESUMO

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.

Documentos Relacionados

• Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli.
• Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination.
• Polarity of heteroduplex formation promoted by Escherichia coli recA protein.
• 3' homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli.
• Escherichia coli RecG and RecA proteins in R-loop formation.