PARP2-QFRD behaves like PARP2-WT in vitro.

A: Cryo-electron micrograph of the PARP2-WT•Nuc165 complex. Enlarged views of two aggregates are shown (1 and 2). Scale bar: 50 nm. B: Thermal denaturation curves of PARP2-WT and PARP2-QFRD from differential scanning fluorimetry. Reported Tm values are the mean and standard deviation from three independent measurements; only one replicate curve is shown for clarity. C: Fluorescence polarization binding curves of PARP2-WT and PARP2-QFRD to fluorescein-labeled p18mer DNA (3 nM). Points and error bars are the mean and standard deviation from three independent measurements (no visible error bar means that the error bar is smaller than the symbol used to plot the data point). Reported KD values are the mean and standard error of the mean. All KD values are listed in Table 1. D: Fluorescence polarization binding curves of PARP2-WT and PARP2-QFRD to Alexa488-labeled Nuc165 (5 nM). Points and error bars are the mean and standard deviation from three independent measurements (no visible error bar means that the error bar is smaller than the symbol used to plot the data point). Reported KD values are the mean and standard error of the mean. All KD values are listed in Table 1. E: Coomassie-stained SDS-PAGE of unmodified and PARylated PARP2-WT and PARP2-QFRD. F: Activation curves of PARP2-WT and PARP2-QFRD (30 nM) by increasing concentrations of p18mer DNA. Points and error bars are the mean and standard deviation from three independent measurements (no error bar means the error bar is smaller than the symbol used to plot the data point). Reported Kact values are the mean and standard error of the mean. G: Fluorescence polarization binding curves of PARP2-WT to fluorescein-labeled p18mer DNA (3 nM) at various NaCl concentrations. KD values at each NaCl concentration tested are listed in S1 Table. H: Fluorescence polarization binding curves of PARP2-QFRD to fluorescein-labeled p18mer DNA (3 nM) at various NaCl concentrations. KD values at each NaCl concentration tested are listed in S1 Table. I: Fluorescence polarization binding curves of PARP2-WT to fluorescein-labeled p18mer DNA (3 nM) in our condition (50 mM NaCl, 1 mM MgCl2) and in an experiment replicating the condition reported by [11] (60 mM KCl, 8 mM MgCl2). Points and error bars are the mean and standard deviation from three independent measurements (no error bar means the error bar is smaller than the symbol used to plot the data point). Reported KD values are the mean and standard error of the mean. J: Fluorescence polarization binding curves of PARP2-QFRD to fluorescein-labeled p18mer DNA (3 nM) in our condition (50 mM NaCl, 1 mM MgCl2) and in an experiment replicating the condition reported by [11] (60 mM KCl, 8 mM MgCl2). Points and error bars are the mean and standard deviation from three independent measurements (no error bar means the error bar is smaller than the symbol used to plot the data point). Reported KD values are the mean and standard error of the mean. K: PARP2 sequence alignment across several species. Q112, F113 are indicated with left-right arrow; WGR domain is labelled as such; Y188 residue is indicated with down arrow. L: Conservation analysis of the WGR domain of PARP2. The Q112 and F113 residues are no more conserved than average. Conserved residues W138, R140 and Y188 are also shown to highlight the contrast in conservation. Protein sequences used in this analysis are listed in Supplementary Information. M: Packing contacts in the crystal lattice of PDB entry 6F5B. DNA is drawn as grey cartoon, WGR domains are shown as surface with one shade of blue for each biological assembly, and the Q112 and F113 residues are colored in red. (RAR)