Asymmetric Regulation of Bipolar Single-stranded DNA Translocation by the Two Motors within Escherichia coli RecBCD Helicase

Repair of double-stranded DNA breaks in Escherichia coli is initiated by the RecBCD helicase that possesses two superfamily-1 motors, RecB (3′ to 5′ translocase) and RecD (5′ to 3′ translocase), that operate on the complementary DNA strands to unwind duplex DNA. However, it is not known whether the RecB and RecD motors act independently or are functionally coupled. Here we show by directly monitoring ATP-driven single-stranded DNA translocation of RecBCD that the 5′ to 3′ rate is always faster than the 3′ to 5′ rate on DNA without a crossover hotspot instigator site and that the translocation rates are coupled asymmetrically. That is, RecB regulates both 3′ to 5′ and 5′ to 3′ translocation, whereas RecD only regulates 5′ to 3′ translocation. We show that the recently identified RecBC secondary translocase activity functions within RecBCD and that this contributes to the coupling. This coupling has implications for how RecBCD activity is regulated after it recognizes a crossover hotspot instigator sequence during DNA unwinding. Background: RecBCD helicase is involved in repair of double-stranded DNA breaks. Results: The 5′ to 3′ ssDNA translocation rate of RecBCD is faster than the 3′ to 5′ rate in the absence of a CHI site, and the rates are coupled asymmetrically. Conclusion: RecBC controls 3′ to 5′ and 5′ to 3′ translocation, but RecD controls only 5′ to 3′ translocation. Significance: Asymmetric regulation may explain how RecBCD is regulated after CHI recognition.