Mechanism of E. coli RecA protein directed strand exchanges in post-replication repair of DNA

Abstract

Escherichia coli mutants carrying recA⁻ are both recombination deficient and unable to perform post-replication repair^1,2. The product of the recA gene regulates the inducible DNA repair functions (the SOS response to DNA damage)^3,4, and is directly involved in homologous pairing^5–8 and strand exchange^9–12, two reactions fundamental to recombination and post-replication repair. The filling of post-replication gaps is thought to occur by homologous pairing of the gapped DNA duplex with an intact duplex, followed by cutting of the intact molecule so that sister strand exchanges can take place. Using in vitro systems, we have shown previously that purified RecA protein binds cooperatively to duplex DNA that contains gaps¹³, and promotes joint molecule formation (synapsis) between gapped and intact duplexes^7,8. Moreover, RecA protein promotes a reciprocal exchange of strands between paired DNA molecules^10,12,14. Here, we investigate the mechanism of sister strand exchange thought to occur during post-replication repair. We show that RecA protein initiates strand exchange from a nicked duplex, transferring the 3′-OH terminus at the nick into the single-stranded (ss) region of the gapped molecule. In the presence of ATP, two heteroduplex molecules are formed as RecA protein drives the reciprocal exchanges in one direction starting at the site of the original crossover.