Chi hotspot control of RecBCD enzyme requires a RecB tether–RecC groove crosspoint interaction

Homologous genetic recombination is required for the faithful repair of broken DNA to continue life and for genetic diversification to propel evolution. The bacterial RecBCD enzyme promotes these processes through coordinated DNA helicase and nuclease activities, which are regulated by a Chi recombination hotspot sequence (5'-GCTGGTGG-3' in enteric bacteria) and the loading of the RecA DNA strand-exchange protein onto the newly generated 3' single-stranded DNA end. Chi's control of RecBCD requires a complex interaction of all three subunits at widely dispersed points in the 330 kDa three-subunit protein. Here, we describe an additional point that is critical for Chi's site-specific stimulation of recombination in Escherichia coli. This point, on the surface of RecBCD, is where the middle of the 19-amino-acid tether connecting the RecB helicase and nuclease domains fits into a groove on the surface of RecC. Deleting or changing even a single amino acid in this crosspoint dramatically reduces Chi hotspot activity. Surprisingly, severing the tether at the RecB helicase junction leaves Chi and RecBCD fully active, but severing the tether at the RecB nuclease junction abolishes Chi activity and strongly reduces recombination proficiency. This difference is accounted for by the critical role of the tether-groove interaction described here. We discuss how Chi controls RecBCD via the coordinated interaction of the tether-groove crosspoint and 13 other widely spaced points throughout RecBCD.