The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non–covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53–PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53–DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome.

Poly(ADP-ribosyl)ation regulates numerous cellular processes like genome maintenance and cell death, thus providing protective functions but also contributing to several pathological conditions. Poly(ADP-ribose) (PAR) molecules exhibit a remarkable heterogeneity in chain lengths and branching frequencies, but the biological significance of this is basically unknown. To unravel structure-specific functions of PAR, we used PARP1 mutants producing PAR of different qualities, i.e. short and hypobranched (PARP1\G972R), short and moderately hyperbranched (PARP1\Y986S), or strongly hyperbranched PAR (PARP1\Y986H). By reconstituting HeLa PARP1 knockout cells, we demonstrate that PARP1\G972R negatively affects cellular endpoints, such as viability, cell cycle progression and genotoxic stress resistance. In contrast, PARP1\Y986S elicits only mild effects, suggesting that PAR branching compensates for short polymer length. Interestingly, PARP1\Y986H exhibits moderate beneficial effects on cell physiology. Furthermore, different PARP1 mutants have distinct effects on molecular processes, such as gene expression and protein localization dynamics of PARP1 itself, and of its downstream factor XRCC1. Finally, the biological relevance of PAR branching is emphasized by the fact that branching frequencies vary considerably during different phases of the DNA damage-induced PARylation reaction and between different mouse tissues. Taken together, this study reveals that PAR branching and chain length essentially affect cellular functions, which further supports the notion of a ‘PAR code’.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD + substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD + and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1dependent PARylation with molecular and temporal resolution.

One of the fastest cellular responses to genotoxic stress is the formation of poly(ADP-ribose) polymers (PAR) by poly(ADP-ribose)polymerase 1 (PARP1, or ARTD1). PARP1 and its enzymatic product PAR regulate diverse biological processes, such as DNA repair, chromatin remodeling, transcription and cell death. However, the inter-dependent function of the PARP1 protein and its enzymatic activity clouds the mechanism underlying the biological response. We generated a PARP1 knock-in mouse model carrying a point mutation in the catalytic domain of PARP1 (D993A), which impairs the kinetics of the PARP1 activity and the PAR chain complexity in vitro and in vivo, designated as hypo-PARylation. PARP1D993A/D993A mice and cells are viable and show no obvious abnormalities. Despite a mild defect in base excision repair (BER), this hypo-PARylation compromises the DNA damage response during DNA replication, leading to cell death or senescence. Strikingly, PARP1D993A/D993A mice are hypersensitive to alkylation in vivo, phenocopying the phenotype of PARP1 knockout mice. Our study thus unravels a novel regulatory mechanism, which could not be revealed by classical loss-of-function studies, on how PAR homeostasis, but not the PARP1 protein, protects cells and organisms from acute DNA damage.

Due to multiple domains and in part intrinsically disordered regions, structural analyses of p53 remain a challenging task, particularly in complex with DNA and other macromolecules. Here, we applied a novel attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic approach to investigate changes in secondary structure of full-length p53 induced by non-covalent interactions with DNA and poly(ADP-ribose) (PAR). To validate our approach, we confirmed a positive regulatory function of p53’s C-terminal domain (CTD) with regard to sequence-specific DNA binding and verified that the CTD mediates p53–PAR interaction. Further, we demonstrate that DNA and PAR interactions result in distinct structural changes of p53, indicating specific binding mechanisms via different domains. A time-dependent analysis of the interplay of DNA and PAR binding to p53 revealed that PAR represents p53’s preferred binding partner, which efficiently controls p53–DNA interaction. Moreover, we provide infrared spectroscopic data on PAR pointing to the absence of regular secondary structural elements. Finally, temperature-induced melting experiments via CD spectroscopy show that DNA binding stabilizes the structure of p53, while PAR binding can shift the irreversible formation of insoluble p53 aggregates to higher temperatures. In conclusion, this study provides detailed insights into the dynamic interplay of p53 binding to DNA and PAR at a formerly inaccessible molecular level.