Probing 3′-ssDNA Loop Formation in E. coli RecBCD/RecBC-DNA Complexes Using Non-natural DNA: A Model for "Chi" Recognition Complexes

The equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends containing varying lengths of polyethylene glycol (PEG) spacers within pre-formed 3′-single-stranded (ss) DNA ((dT)_n) tails was studied. These studies were designed to test a previous proposal that the 3′-(dT)_n tail can be looped out upon binding RecBC and RecBCD for 3′-ssDNA tails with n ≥ 6 nucleotides. Equilibrium binding of protein to unlabeled DNA substrates with ends containing PEG-substituted 3′-ssDNA tails was examined by competition with a Cy3-labeled reference DNA which undergoes a Cy3 fluorescence enhancement upon protein binding. We find that the binding affinities of both RecBC and RecBCD for a DNA end are unaffected upon substituting PEG for the ssDNA between the sixth and the final two nucleotides of the 3′-(dT)_n tail. However, placing PEG at the end of the 3′-(dT)_n tail increases the binding affinities to their maximum values (i.e. the same as binding constants for RecBC or RecBCD to a DNA end with only a 3′-(dT)₆ tail). Equilibrium binding studies of a RecBC mutant containing a nuclease domain deletion, RecB^ΔnucC, suggest that looping of the 3′-tail (when n ≥ 6 nucleotides) occurs even in the absence of the RecB nuclease domain, although the nuclease domain stabilizes such loop formation. Computer modeling of the RecBCD-DNA complexes suggests that the loop in the 3′-ssDNA tail may form at the RecB/RecC interface. Based on these results we suggest a model for how a loop in the 3′-ssDNA tail might form upon encounter of a "Chi" recognition sequence during unwinding of DNA by the RecBCD helicase.