Code used in MATLAB and R for the purpose of generating HX difference plots and HX ribbon plots

PARP1 and PARP2 recognize DNA breaks immediately upon their formation, generate a burst of local PARylation to signal their location, and are co-targeted by all current FDA-approved forms of PARP inhibitors (PARPi) used in the cancer clinic. Recent evidence indicates that the same PARPi molecules impact PARP2 differently from PARP1, raising the possibility that allosteric activation may also differ. We find that unlike for PARP1, destabilization of the autoinhibitory domain of PARP2 is insufficient for DNA damage-induced catalytic activation. Rather, PARP2 activation requires further unfolding of an active site helix. In contrast, the corresponding helix in PARP1 only transiently forms, even prior to engaging DNA. Only one clinical PARPi, Olaparib, stabilizes the PARP2 active site helix, representing a structural feature with the potential to discriminate small molecule inhibitors. Collectively, our findings reveal unanticipated differences in local structure and changes in activation-coupled backbone dynamics between human PARP1 and PARP2.

Methods
HDExaminer software (v 2.5.0) was used, which uses peptide pool information to identify the deuterated peptides for every sample in the HXMS experiment. The quality of each peptide was further assessed by manually checking mass spectra. The level of HX of each reported deuterated peptide is corrected for loss of deuterium label (back-exchange after quench) during HXMS data collection by normalizing to the maximal deuteration level of that peptide in the fully-deuterated (FD) samples. After normalizing, we then compared the extent of deuteration to the theoretical maximal deuteration (maxD, i.e. if no back-exchange occurs). The data analysis statistics for all the protein states are in Table S2 of Smith-Pillet et al., Mol cell 2025. The difference plots for the deuteration levels between any two samples were obtained through an in-house script written in MATLAB. The script compares the deuteration levels between two samples (e.g. PARP2 and PARP2 with 5’P nicked DNA) and plots the percent difference of each peptide, by subtracting the percent deuteration of PARP2 with 5’P nicked DNA from PARP2 and plotting according to the color legend in stepwise increments. The plot of representative peptide data is shown as the mean of three independent measurements +/- SD. Statistical analysis included a t-test with a P-value <0.05. HX experiments of PARP1 with or without DNA and/or EB-47 have been published. To compare PARP1 and PARP2 datasets, HX samples of PARP1 were repeated in triplicate to have the same peptide digestions and subsequent peptide data, and HX changes in HD peptides were compared between PARP1 and PARP2 with the indicated conditions. HXMS data at 100 s for PARP2 and in the presence of gap DNA, 5’OH nicked DNA, and 5’P nicked DNA was plotted through an in-house script written in R (see Fig. S1A in Smith-Pillet et al., Mol cell 2025).