Mechanistic Insights into the Stimulation of Arabidopsis PARP2 Self-Parylation Catalytic Activity by DNA Breaks and...

Poly(ADP-ribose) polymerases (PARPs) play a central role in maintaining genome stability and catalyzing the transfer of ADP-ribose units from NAD⁺ to target proteins through a process known as poly(ADP-ribosyl)ation (PARylation). While mammalian PARPs are extensively characterized, their plant homologues remain less explored. Here, we dissect the mechanistic regulation of Arabidopsis thaliana PARP2 (atPARP2) through its interactions with DNA and poly(ADP-ribose) (PAR). Biolayer interferometry (BLI) binding studies revealed that atPARP2 recognizes phosphorylated DNA double-strand breaks (p-DSBs) and PAR with a nanomolar affinity. Using the SAP domain of atPARP2, we reported for the first time that it binds both PAR and p-DSBs, thereby significantly enhancing the binding affinity of atPARP2 for PAR and p-DSBs. In addition, isothermal titration calorimetry (ITC) and biochemical assays demonstrated that PAR serves as an allosteric activator of atPARP2, with the WGR domain acting as a key module for PAR binding. Enzymatic assays further established the indispensable role of SAP domains in enhancing DNA- and PAR-dependent catalytic activities. Together, our findings demonstrate that atPARP2 may employ a modular strategy, integrating DNA break sensing and PAR recognition, to fine-tune its catalytic function in response to genotoxic stress.