The Nuclease Domain of E. coli RecBCD Regulates DNA Binding and Base Pair Melting

Abstract E. coli RecBCD, a hetero-trimeric helicase and nuclease, functions in double stranded (ds) DNA break repair and in degrading foreign DNA. RecBCD possesses ATPase motor domains within both RecB (3’ to 5’) and RecD (5’ to 3’) and a nuclease domain within RecB (RecBNuc). RecBCD binds to double stranded DNA ends and initiates DNA unwinding by first melting several DNA base pairs (bp) using only its binding free energy. The RecBNuc domain is docked ∼70 Å from the duplex DNA binding site in RecBCD-DNA structures but appears to be dynamic and able to move from its docked position. Here, we compare DNA binding of RecBCD and a variant, RecBΔNucCD, in which the 30 kDa nuclease domain has been deleted. RecBCD binding to a blunt DNA end is enthalpically unfavorable and entropically driven. Deletion of RecBNuc results in an increase in DNA binding affinity, suggesting an allosteric effect of RecBNuc. RecBΔNucCD binding to DNA possessing fully ‘pre-melted’ DNA ends is associated with a large favorable ΔHobs, but much smaller than observed for RecBCD, suggesting that deletion of RecBNuc limits bp melting from a blunt DNA. We also solved cryo-EM structures showing only 4 bp melted upon RecBΔNucCD binding to a blunt ended DNA duplex, less than the 11 bp melted upon RecBCD binding. Thus, the RecB nuclease domain regulates the extent of bp melting by RecBCD. These results also suggest that RecBNuc may manifest its long-range allosteric effect on DNA binding and DNA melting via linker-linker interactions between RecB and RecC. Competing Interest Statement The authors have declared no competing interest.