Activation, interactions, and inhibition of ADP- ribosyltransferase PARP2 in DNA damage repair

ADP-ribosylation is a post-translational modification that plays a critical role in the DNA damage response. In humans, 22 ADP-ribosyltransferases have been identified, among which PARP1, PARP2, and PARP3 are activated by DNA damage. In response to DNA damage, these enzymes catalyse ADPribosylation by transferring ADP-ribosyl moieties to target protein and themselves. PARP1/2/3 share conserved C-terminal domains responsible for catalytic activity, whereas their N-terminal regions are highly divergent and likely contribute towards DNA damage the recognition and mediate enzyme activation. In this thesis, we investigated the role of the PARP2 N-terminus in its automodification and release from DNA. We identified key serine residues involved in automodification using a gel-based PARylation assay, and examined their impact on PARP2 release from DNA using a fluorescence polarization assay. Our results demonstrate that, in the presence of HPF1, Ser8 and Ser73 at the N-terminus of PARP2 are primary sites of ADP-ribosylation, with Ser8 contributing to the majority of PARP2 automodification. These findings highlight the mechanistic importance of N-terminus of the PARP2 in ADP-ribosylationdependent release from DNA. The ADP-ribosylation plays an important role in DNA damage, and serine ADP-ribosylation promotes the recruitment of chromatin modellers to facilitate DNA repair. Serine ADP-ribosylation results from the interaction between PARP1/2 and HPF1. Therefore, targeting the PARP1/2-HPF1 interaction represents an alternative strategy to modulate serine ADP-ribosylation. To this end, we developed a FRET-based high-throughput assay for screening compound libraries for inhibitors of the PARP-HPF1 interaction. Using this assay, we identified Alkannin and Dimethylacrylshikonin as hit compounds that disrupt the PARP-HPF1 interaction. Importantly, these compounds inhibited the ADP-ribosylation activity of full-length PARP1 and PARP2 by disrupting PARP-HPF1 complex formation. Overall, this thesis work provides insights into the involvement of the PARP2 N-terminus in its activity and introduces a robust screening platform for the discovery of inhibitors targeting the PARP–HPF1 interaction.