Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sequential eviction of crowded nucleoprotein complexes by the exonuclease RecBCD molecular motor

doi: 10.1073/pnas.1701368114

Figure Lengend Snippet: Engineered 5×, 10×, 30×, and 50× arrays of EcoRI binding sites on λ-DNA. (A) A schematic of the λ-DNA and a schematic of the expected fragment sizes for the different DNA array lengths when digested with SalI and EagI. (B) A 1% agarose gel showing that the length of engineered sites is as expected. In this assay, 5-μL aliquots of the engineered λ-DNA were incubated with 1 μL of SalI-HF (R3138S; New England BioLabs) and EagI-HF (R3505S; New England BioLabs) in 38 μL water supplemented with 5 μL of 5× CutSmart buffer (B7204S; New England BioLabs) for 2 h. The reaction was mixed with 10 μL of 6× loading dye (B7024S; New England BioLabs) and run on the gel for 1 h at 100 V; a 1-kb DNA ladder (N3232S; New England BioLabs) was also run. The magenta arrow highlights the 3.3-kb native DNA fragment [SalI (33.4 kb) to EagI (36.7 kb)] present before cloning in the array fragments. The green arrows highlight the array-containing fragments for each of the different substrates. MW, molecular weight.

Article Snippet: In this assay, 5-μL aliquots of the engineered λ-DNA were incubated with 1 μL of SalI-HF (R3138S; New England BioLabs) and EagI-HF (R3505S; New England BioLabs) in 38 μL water supplemented with 5 μL of 5× CutSmart buffer (B7204S; New England BioLabs) for 2 h. The reaction was mixed with 10 μL of 6× loading dye (B7024S; New England BioLabs) and run on the gel for 1 h at 100 V; a 1-kb DNA ladder (N3232S; New England BioLabs) was also run.

Techniques: Binding Assay, DNA Array, Agarose Gel Electrophoresis, Incubation, Clone Assay, Molecular Weight

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sequential eviction of crowded nucleoprotein complexes by the exonuclease RecBCD molecular motor

doi: 10.1073/pnas.1701368114

Figure Lengend Snippet: The saturation of the λ-DNA substrate with EcoRIE111Q. (A) An example of the bulk biochemical assay used to establish conditions for saturating the EcoRI binding site arrays with EcoRIE111Q. In this example, the λ-DNA substrate bearing a 5× binding site array was preincubated with varying concentrations of EcoRIE111Q for 30 min at room temperature. The reactions were then challenged by the addition of WT EcoRI for 15 min at 37 °C. DNA products were then resolved on a 1% agarose gel. (B) Position distribution histogram from a DNA curtain assay with Qdot-tagged EcoRIE111Q bound to a λ-DNA harboring a 5× array. The location of the array is indicated along with the four peaks corresponding to the native EcoRI binding sites. The height of the 5× peak is similar to the 1× peak heights, because the data are not corrected for signal intensity.

Article Snippet: In this assay, 5-μL aliquots of the engineered λ-DNA were incubated with 1 μL of SalI-HF (R3138S; New England BioLabs) and EagI-HF (R3505S; New England BioLabs) in 38 μL water supplemented with 5 μL of 5× CutSmart buffer (B7204S; New England BioLabs) for 2 h. The reaction was mixed with 10 μL of 6× loading dye (B7024S; New England BioLabs) and run on the gel for 1 h at 100 V; a 1-kb DNA ladder (N3232S; New England BioLabs) was also run.

Techniques: Binding Assay, Agarose Gel Electrophoresis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sequential eviction of crowded nucleoprotein complexes by the exonuclease RecBCD molecular motor

doi: 10.1073/pnas.1701368114

Figure Lengend Snippet: Sequential eviction of EcoRI from DNA by RecBCD. (A) A model describing the predicted outcome for two-color labeling experiments designed to test for sequential protein eviction by RecBCD; details are presented in the text. (B) Examples of two-color kymographs showing Qdot-tagged EcoRIE111Q (Qdot 705; magenta) being pushed into a 5× array bound by Qdot-tagged EcoRIE111Q (Qdot 605; green). Reactions were initiated by addition of 1 mM ATP into the RecBCD buffer (40 mM Tris⋅HCl, pH 7.5, 2 mM MgCl2, 0.2 mg mL–1 Pluronic). Gaps in the EcoRIE111Q traces result from Qdot blinking. (C) Schematic illustration of the experiment used to assess the fate of EcoRIE111Q bound to native target sites in the λ-DNA located either upstream or downstream of the 5× EcoRI array (Upper) and the resulting dissociation positions of the upstream and downstream EcoRIE111Q molecules (Lower). (D) Survival probability plots for EcoRIE111Q molecules located upstream and downstream of the 5× EcoRI arrays. The horizontal axis represents the distance from the original EcoRI binding position. The error bars represent SDs calculated by bootstrap analysis.

Article Snippet: In this assay, 5-μL aliquots of the engineered λ-DNA were incubated with 1 μL of SalI-HF (R3138S; New England BioLabs) and EagI-HF (R3505S; New England BioLabs) in 38 μL water supplemented with 5 μL of 5× CutSmart buffer (B7204S; New England BioLabs) for 2 h. The reaction was mixed with 10 μL of 6× loading dye (B7024S; New England BioLabs) and run on the gel for 1 h at 100 V; a 1-kb DNA ladder (N3232S; New England BioLabs) was also run.

Techniques: Labeling, Binding Assay