Probing 3 '-ssDNA loop formation in E-coli RecBCD/RecBC-DNA complexes using non-natural DNA: A model for 'Chi' recognition complexes

摘要

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(Delta nuc)C, 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. (c) 2006 Elsevier Ltd. All rights reserved.