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. 2021 Nov 18;12:6675. doi: 10.1038/s41467-021-27043-8

Fig. 6. HPF1 further destabilizes the HD when PARP1 is bound to a DNA SSB.

Fig. 6

a An HXMS difference plot obtained by subtracting the percent deuteration of the PARP1/DNA/HPF1 complex (at 1:1 ratio of HPF1:PARP1) from PARP-1/DNA complex at 10 s. Each horizontal bar represents a PARP1 peptide. Most of the peptides (gray) in the PARP1/DNA/HPF1 complex have similar HX rates when compared to the PARP1/DNA complex. However, peptides in the αB and αF helices of HD showed faster exchange (red). The white regions in the difference plot represent gaps in the peptide coverage. b HX of a representative peptide from the αB helix in panel a for PARP1/DNA and PARP1/DNA/HPF1. c HX of a representative peptide from the αF helix in panel a for PARP1/DNA and PARP1/DNA/HPF1. d HX of representative peptide from WGR domain in panel a for PARP1/DNA and PARP1/DNA/HPF1. In panels b to d, an average is shown with error bars representing SD and asterisks indicating P < 0.05 (t-test two-sided between PARP1/DNA and PARP1/DNA/HPF1). For αB at 10 s, P = 0.0003, at 100 s, P = 0.0121. For αF at 10 s, P = 0.0018, at 100 s, P = 0.0073. Experiments were performed in triplicate. In panels b–d, the blue dotted lines indicate the maximum number of exchangeable deuterons (maxD). e Consensus HXMS data from panel a mapped onto the structure of the catalytic domain of PARP1 (from 4DQY), which was modeled in complex with HPF1, based on the PARP1CATΔHD complex with HPF1 (6TX3). f DNA binding affinity of PARP1 in the absence and presence of HPF1 determined by fluorescence polarization. An average of six independent experiments for PARP1 WT and PARP1 R865A, four independent experiments for PARP1 WT with HPF1, and three for PARP1 R865A with HPF1 is shown with associated SD represented by error bars. Two-sample two-sided t-tests were used to compare the KD values and asterisks indicate P < 0.05. ns, indicates not significant. The P-values are 0.014 for WT and WT + HPF1, 0.000039 for WT and R865A, and 0.087 for R865A and PARP1 R865A + HPF1. Source data are provided as a Source Data file.