2 deoxy atp

ATP and ADP were prepared fresh from powder and were purchased from Sigma. ATP and ADP concentrations were determined by absorbance at 259 nm using ε259 of 15,400 M^–1⋅cm^–1. N-Methylanthraniloyl (mant)-labeled 2′-deoxy-ADP and 2′-deoxy-ATP were purchased from Jena Biosciences. The mantATP and mantADP concentrations were determined from absorbance measurements at 255 nm using ε255 of 23,300 M^–1⋅cm^–1. Nucleotides were prepared prior to use in the presence of equimolar MgCl₂.

All chemicals and reagents were the highest purity commercially available. ATP and ADP were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). Adenosine 5′-(β,γ-imido)triphosphate (AMPpNp) was purchased from Sigma (St. Louis, MO, USA). A molar equivalent of MgCl₂ was added to nucleotides immediately before use. Nucleotide concentrations were determined by absorbance using an extinction coefficient e₂₅₉ of 15 400 M⁻¹ cm⁻¹. The concentrations of N-methylanthraniloyl (mant) derivatives of ADP, 2′-deoxyADP, ATP, and 2′-deoxyATP (Jena Bioscience, Jena, Germany) were determined using e₂₅₅ of 23 300 M⁻¹ cm⁻¹. Unless otherwise specified, all experiments were conducted in RecBCD Buffer (RB: 20 mM MOPS pH 7.4, 75 mM NaCl, 2 mM MgCl₂, 1 mM DTT). Over-expression and purification of recombinant WT RecBCD and mutant RecB^K29QC were based on the previously described method (33 (link),34 (link)), with an additional step described by Zananiri et al. (35 (link)). The RecBCD concentration was determined using e₂₈₀ = 4.2 × 10⁵ M⁻¹ cm⁻¹ in guanidinium chloride. RecBCD purity of nucleic acid contaminants was determined by measuring the absorption ratio of 280/260 nm, and only protein fractions with a ratio >1.3 were used.