The Translocating RecBCD Enzyme Stimulates Recombination by Directing RecA Protein onto ssDNA in a χ-Regulated Manner

1994). The RecBCD enzyme binds the dsDNA breaks Double-stranded DNA break repair and homologous with a high affinity and then proceeds to unwind while recombination in E. coli are initiated by the RecBCD simultaneously degrading the DNA (Telander-Muskaenzyme, which unwinds and simultaneously degrades vitch and Linn, 1981; Taylor and Smith, 1985). This degDNA from a double-stranded DNA end. This process radation is asymmetric, with the 39-terminal strand at is stimulated by cis-acting DNA elements, known as the entry site for RecBCD enzyme being degraded much x sites. Using both in vitropairing and nuclease protecmore extensively than the 59-terminal strand (Dixon and tion assays, we demonstrate that the translocating Kowalczykowski, 1993). One interesting aspect of this RecBCD enzyme, which has been activated by x, coorrecombination pathway is that it is stimulated by the dinates the preferential loading of the homologous presence of DNA sequence elements known as x sites pairingprotein, RecA, onto theresultant single-strand(Lam et al., 1974; Stahl et al., 1975; Smith et al., 1981). ed DNA downstream of x. This facilitated loading of The x site (59-GCTGGTGG-39) stimulates recombination RecA protein results in a substantial increase in both 5to 10-fold in its vicinity, and this stimulation is detectthe efficiency and rate of in vitro recombination reacable up to 10 kb downstream of the x site (Stahl et al., tions and offers an explanation for stimulation of re1980; Ennis et al., 1987; Cheng and Smith, 1989; Myers combination and repair in vivo by x. et al., 1995). Upon encountering a properly oriented x