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Figure EV2. Characterization of mutant XPF‐ERCC1 complexes A. Superdex 200 gel filtration column elution profile of XPFL219R‐ERCC1. The heterodimeric fraction depicted in Fig 1A was collected, concentrated, and rerun on the same column. The protein did not aggregate and eluted as a heterodimer at ˜65 ml. B. Replicate of Fig 1D. 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. C. As in (B) but using a 5′‐FAM‐labeled 3′ flap DNA substrate. D. As in (B) but using higher concentrations of the XER670S mutant. E. Replicate of Fig 1E. 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. 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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