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. 2017 Jun 12;36(14):2047–2060. doi: 10.15252/embj.201796664

Figure EV4. RPA stimulation of XPF‐ERCC1 activity on “+leading‐strand” structure is not attributed to the displacement of the model nascent leading strand or the unwinding of the fork substrates by RPA.

Figure EV4

  1. Nuclease activity of XE on the indicated fork substrates in the presence or absence of RPA. RPA specifically stimulates XE activity on a “simple fork” and “+leading‐strand” substrates.
  2. Fluorescence anisotropy assay to determine the binding constants of RPA for either “simple fork” or “+lagging‐strand” substrates. The blue diamonds denote the fluorophore‐labelled nucleotides. Error bars represent SD, n = 3.
  3. (Top panel) Outline of potential consequences of incubating “+leading‐strand” substrate radiolabelled on the model nascent leading strand with RPA, and the potential products that might be revealed by analysis on a non‐denaturing PAGE gel. (Bottom panel) Nuclease activity performed as in panel a. Reaction products were separated on a 10% non‐denaturing PAGE gel. The DNA substrates remain intact in the RPA alone reactions (lanes 4 and 9), indicating that RPA does not displace the model nascent leading strand or unwind the fork substrates, at the concentrations employed.

Source data are available online for this figure.