RecBCD Enzyme Switches Lead Motor Subunits in Response to χ Recognition

SummaryRecBCD is a DNA helicase comprising two motor subunits, RecB and RecD. Recognition of the recombination hotspot, χ, causes RecBCD to pause and reduce translocation speed. To understand this control of translocation, we used single-molecule visualization to compare RecBCD to the RecBCDK177Q mutant with a defective RecD motor. RecBCDK177Q paused at χ but did not change its translocation velocity. RecBCDK177Q translocated at the same rate as the wild-type post-χ enzyme, implicating RecB as the lead motor after χ. P1 nuclease treatment eliminated the wild-type enzyme's velocity changes, revealing a χ-containing ssDNA loop preceding χ recognition and showing that RecD is the faster motor before χ. We conclude that before χ, RecD is the lead motor but after χ, the slower RecB motor leads, implying a switch in motors at χ. We suggest that degradation of foreign DNA needs fast translocation, whereas DNA repair uses slower translocation to coordinate RecA loading onto ssDNA.