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. 2021 Dec 22;601(7892):268–273. doi: 10.1038/s41586-021-04261-0

Fig. 3. HELQ exhibits sequence-independent RPA-coated DNA capture activity.

Fig. 3

a, Schematics of the experimental optical tweezer set-up for observing the capture of Cy3-labelled DNA oligos. b, Kymographs showing the capture of targeted Cy3-labelled 80-mer oligo (λ4) in trans with HELQ and HELQ(K365M) in the presence of RPA–eGFP at multiple positions of RPA–eGFP-coated λ-ssDNA (top). KM, HELQ(K365M). Kymographs showing the capture of non-targeted Cy3-labelled dT 79 homopolymer in trans with HELQ in the presence of RPA–eGFP at multiple positions of RPA–eGFP-coated λ-ssDNA (bottom). Scale bars, 60 s (horizontal), 10 µm (vertical). c, Quantification of the experiments shown in b. Each datapoint represents a single DNA molecule. Data are mean ± s.d. Statistical analysis was performed using two-sided Mann–Whitney U-tests. No adjustments were made for multiple comparisons. d, The dwell times of captured λ4 by HELQ in the presence of RPA–eGFP. n = 61. The black line represents the exponential fit. Tau = 134 s. e, The dwell times of captured λ4 by HELQ(K365M) in the presence of RPA–eGFP. n = 87. The black line represents the exponential fit. Tau = 179 s. f, The experimental conditions for the optical tweezer experiment in g. g, Kymograph showing the Cy3–λ4 oligo captured on RPA–eGFP-coated λ-ssDNA in the presence of HELQ and RPA–eGFP after stretching of tethered λ-ssDNA by a gradual increase of force. Scale bars, 60 s (horizontal), 10 µm (vertical). h, Schematic of the bulk capture assay. i, Native gel showing the capture assay with the indicated concentrations of HELQ and RPA (82 nM). B indicates biotin at the 3′ end of B-dT43 oligo. The experiment was performed twice with similar results.