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

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• 作者：C. Jason Wong ; Rachel L. Rice ; Nathan A. Baker ; Tao Ju and Timothy M. Lohman
• 关键词：helicase ; fluorescence ; motor protein ; recombination ; thermodynamics
• 刊名：Journal of Molecular Biology
• 出版年：2006
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：8 September 2006
• 卷：362
• 期：1
• 页码：26-43
• 文件大小：973 K

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^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 ȁc;Chi” recognition sequence during unwinding of DNA by the RecBCD helicase.