Reber, Julia M.
PARP1 and XRCC1 exhibit a reciprocal relationship in genotoxic stress response
2023, Reber, Julia M., Božić-Petković, Jovana, Lippmann, Michelle, Mazzardo, Marvin, Dilger, Asisa, Warmers, Rebecca, Bürkle, Alexander, Mangerich, Aswin
PARP1 (aka ARTD1) acts as a prime sensor of cellular genotoxic stress response. PARP1 detects DNA strand breaks and subsequently catalyzes the formation of poly(ADP-ribose) (PAR), which leads to the recruitment of the scaffold protein XRCC1 during base excision and single strand break repair and the assembly of multi-protein complexes to promote DNA repair. Here, we reveal that the recruitment of either protein to sites of DNA damage is impeded in the absence of the other, indicating a strong reciprocal relationship between the two DNA repair factors during genotoxic stress response. We further analyzed several cellular and molecular endpoints in HeLa PARP1 KO, XRCC1 KO, and PARP1/XRCC1 double KO (DKO) cells after genotoxic treatments, i.e., PARylation response, NAD+ levels, clonogenic survival, cell cycle progression, cell death, and DNA repair. The analysis of NAD+ levels and cytotoxicity after treatment with the topoisomerase I inhibitor camptothecin revealed a hypersensitivity phenotype of XRCC1 KO cells compared to PARP1 KO cells-an effect that could be rescued by the additional genetic deletion of PARP1 as well as by pharmacological PARP inhibition. Moreover, impaired repair of hydrogen peroxide and CPT-induced DNA damage in XRCC1 KO cells could be partially rescued by additional deletion of PARP1. Our results therefore highlight important reciprocal regulatory functions of XRCC1 and PARP1 during genotoxic stress response.
Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease
2021-06, Prokhorova, Evgeniia, Agnew, Thomas, Wondisford, Anne R., Tellier, Michael, Kaminski, Nicole, Beijer, Danique, Reber, Julia M., Krassnig, Sarah C., Mangerich, Aswin, Ahel, Ivan
ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.
Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal
2021-12, Rack, Johannes Gregor Matthias, Liu, Qiang, Zorzini, Valentina, Voorneveld, Jim, Ariza, Antonio, Reber, Julia M., Krassnig, Sarah C., Mangerich, Aswin, Filippov, Dmitri V., Ahel, Ivan
Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.
At the crossroads of ADP-ribose, PARP1, and XRCC1 in the genotoxic stress response
2021, Reber, Julia M.
ADP-ribosylation is ADP-ribosylation is an essential post-translational modification that plays key roles in a multitude of (patho-) physiological processes and is, among others, catalysed by members of the poly(ADP-ribose) polymerase (PARP) family. PARPs use nicotinamide adenine dinucleotide (NAD+) as a substrate to attach ADP-ribose moieties to target molecules, ranging from DNA and RNA to a vast number of proteins that can be modified at various amino acids. Modifications can either consist of a single ADP-ribose unit (i.e. mono-ADP-ribosylation, MARylation) or extend up to 200 units in length, adopting linear and/or branched conformations (i.e. poly(ADP-ribosyl)ation, PARylation). Besides covalent modification, proteins can also non-covalently interact with PAR chains through various PAR reader modules. Collectively, ADP-ribosylation is a highly diverse modification and its dynamic is not only contingent on PAR formation, but also on various catabolising enzymes that swiftly remove ADP-ribose moieties or PAR chains from target molecules. Although PARylation has been extensively studied in recent years, several open questions remain, and the overall goal of this thesis was to further elucidate the biological significance of PAR structural diversity, the interplay between the two important DNA repair factors PARP1 and XRCC1, and finally the interplay between the main PAR catabolising enzymes PARG and ARH3.
Why structure and chain length matter : on the biological significance underlying the structural heterogeneity of poly(ADP-ribose)
2021-09-07, Reber, Julia M., Mangerich, Aswin
Poly(ADP-ribosyl)ation (PARylation) is a multifaceted post-translational modification, carried out by poly(ADP-ribosyl)transferases (poly-ARTs, PARPs), which play essential roles in (patho-) physiology, as well as cancer therapy. Using NAD+ as a substrate, acceptors, such as proteins and nucleic acids, can be modified with either single ADP-ribose units or polymers, varying considerably in length and branching. Recently, the importance of PAR structural heterogeneity with regards to chain length and branching came into focus. Here, we provide a concise overview on the current knowledge of the biochemical and physiological significance of such differently structured PAR. There is increasing evidence revealing that PAR’s structural diversity influences the binding characteristics of its readers, PAR catabolism, and the dynamics of biomolecular condensates. Thereby, it shapes various cellular processes, such as DNA damage response and cell cycle regulation. Contrary to the knowledge on the consequences of PAR’s structural diversity, insight into its determinants is just emerging, pointing to specific roles of different PARP members and accessory factors. In the future, it will be interesting to study the interplay with other post-translational modifications, the contribution of natural PARP variants, and the regulatory role of accessory molecules. This has the exciting potential for new therapeutic approaches, with the targeted modulation and tuning of PARPs’ enzymatic functions, rather than their complete inhibition, as a central premise.
PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response
2020-10-09, Aberle, Lisa, Krüger, Annika, Reber, Julia M., Lippmann, Michelle, Schmalz, Michael, Trussina, Irmela R.E.A., Schlesiger, Sarah, Zubel, Tabea, Schütz, Karina, Marx, Andreas, Ferrando-May, Elisa, Bürkle, Alexander, Mangerich, Aswin
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'.