Homologous pairing in vitro stimulated by the recombination Hotspot, Chi

Genetic recombination in Escherichia coli is stimulated at DNA sequences known as Chi sites, 5′-GCT-GGTGG-3′. We describe the in vitro formation of homologously paired joint molecules that is dependent upon this recombination hotspot. Chi-dependent joint molecule formation requires RecA, RecBCD, and SSB proteins and a Chi site in the donor linear dsDNA. The donor dsDNA is unwound by RecBCD enzyme, and the invasive strand is generated by nicking at Chi. This Chi-dependent invading strand must contain homology to the recipient supercoiled DNA substrate at its newly formed 3′ end for efficient joint molecule formation. Action at Chi generates invasive ssDNA from the 5′ but not the 3′ side of Chi, suggesting that the nuclease activity of RecBCD enzyme is attenuated upon encountering a Chi site. These results support the view that RecBCD enzyme action can precede RecA protein action and reconcile the seemingly opposing degradative and recombination functions of RecBCD enzyme.