Energetics of DNA End Binding by E. coli RecBC and RecBCD Helicases Indicate Loop Formation in the 3′-Single-stranded DNA Tail

We examined the equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends possessing pre-existing single-stranded (ss) DNA ((dT)n) tails varying in length (n=0 to 20 nucleotides) in order to determine the contributions of both the 3′ and 5′ single strands to the energetics of complex formation. Protein binding was monitored by the fluorescence enhancement of a reference DNA labeled at its end with a Cy3 fluorophore. Binding to unlabeled DNA was examined by competition titrations with the Cy3-labeled reference DNA. The affinities of both RecBC and RecBCD increase as the 3′-(dT)n tail length increases from zero to six nucleotides, but then decrease dramatically as the 3′-(dT)n tail length increases from six to 20 nucleotides. Isothermal titration calorimetry experiments with RecBC show that the binding enthalpy is negative and increases in magnitude with increasing 3′-(dT)n tail length up to n=6 nucleotides, but remains constant for n ≥6. Hence, the decrease in binding affinity for 3′-(dT)n tail lengths with n ≥6 is due to an unfavorable entropic contribution. RecBC binds optimally to duplex DNA with (dT)6 tails on both the 3′ and 5′-ends while RecBCD prefers duplex DNA with 3′-(dT)6 and 5′-(dT)10 tails. These data suggest that both RecBC and RecBCD helicases can destabilize or “melt out” six base-pairs upon binding to a blunt DNA duplex end in the absence of ATP. These results also provide the first evidence that a loop in the 3′-ssDNA tail can form upon binding of RecBC or RecBCD with DNA duplexes containing a pre-formed 3′-ssDNA tail with n ≥6 nucleotides. Such loops may be representative of those hypothesized to form upon interaction of a Chi site contained within the unwound 3′ ss-DNA tail with the RecC subunit during DNA unwinding.