Xenbase Image ID: 156408

Fig. 1. Characterization of mutant XPF‐ERCC1 complexes. A. Schematic representation of the domain organization of the XPF protein. Domain boundaries of human and Xenopus laevis XPF are indicated. Relevant mutations of the human protein, and the Xenopus laevis equivalents, are indicated on top and bottom, respectively. B. Superdex 200 gel filtration column elution profile of wild‐type XPF‐ERCC1 and indicated mutant complexes. Aggregates eluted in the void volume of the column (˜45 ml) while the active XPF‐ERCC1 heterodimer eluted at ˜65 ml. The peak eluting at ˜105 ml contains the FLAG peptide used to elute the protein from the FLAG affinity resin. The heterodimer peak was isolated, and proteins were separated on SDS–PAGE and stained with Coomassie blue (inset). C. As in (B) but for different mutant complexes that showed more aggregation. D. Wild‐type and indicated mutant XPF‐ERCC1 complexes were incubated with a 5′‐FAM‐labeled stem‐loop DNA substrate (10 nM) at room temperature for 30 min. Reaction products were separated on a 12% urea–PAGE gel and visualized using a fluorescence imaging system. Red arrow indicates position of incision by XPF‐ERCC1. E. Wild‐type and mutant XPF‐ERCC1 complexes at various concentrations were incubated with a 5′‐FAM‐labeled 3′ flap DNA substrate (10 nM) and fluorescent anisotropy was measured. Graphs were fitted to calculate dissociation constants (Kds) as described in the Materials and Methods section. The error bars represent s.d. from three measurements. Experimental replicates are shown in Fig EV2. Source data are available online for this figure. Image published in: Klein Douwel D et al. (2017) © 2017 The Authors. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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