title: Probing 3'-ssDNA loop formation in E. coli RecBCD/RecBC-DNA complexes using non-natural DNA: a model for "Chi" recognition complexes.
abstract: 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>or=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(Deltanuc)C, suggest that looping of the 3'-tail (when n>or=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.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, Box 8231, St Louis, MO 63110-1093, USA.
authors.forename: C Jason
authors.initials: CJ
authors.lastname: Wong
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Rice
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Baker
authors.forename: Tao
authors.initials: T
authors.lastname: Ju
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Lohman
mesh_terms: D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013816:Thermodynamics
chemical_list: D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli