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.

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure–function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1–PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\L713F expression triggered apoptosis, whereas PARP1\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure–function relationships of natural and artificial PARP1 variants.

DNA replication stress is a major source of DNA strand breaks and genomic instability, and a hallmark of precancerous lesions. In these hyperproliferative tissues, activation of the DNA damage response results in apoptosis or senescence preventing or delaying their development to full malignancy. In cells, in which this antitumor barrier is disabled by mutations (for example, in p53), viability and further uncontrolled proliferation depend on factors that help to cope with replication-associated DNA damage. Replication problems preferentially arise in chromatin regions harboring complex DNA structures. DEK is a unique chromatin architectural factor which binds to non-B-form DNA structures, such as cruciform DNA or four-way junctions. It regulates DNA topology and chromatin organization, and is essential for the maintenance of heterochromatin integrity. Since its isolation as part of an oncogenic fusion in a subtype of AML, DEK has been consistently associated with tumor progression and chemoresistance. How DEK promotes cancer, however, is poorly understood. Here we show that DEK facilitates cellular proliferation under conditions of DNA replication stress by promoting replication fork progression. DEK also protects from the transmission of DNA damage to the daughter cell generation. We propose that DEK counteracts replication stress and ensures proliferative advantage by resolving problematic DNA and/or chromatin structures at the replication fork. INTRODUC110NDEK is a biochemically and structurally unique non-histone chromatin protein which is conserved in all higher eukaryotes. It binds to non-8-form DNA structures, such as cruciform DNA or four-way junctions, regulates DNA topology and chromatin organization, and is essential for the maintenance of heterochromatin integrity. •2 At the cellular level, it displays pleiotropic functions and has been shown to influence differentiation, apoptosis, senescence and maintenance of cell stemness. -6Since its isolation as part of an oncogenic fusion in a subtype of acute myeloid leukemia, 7 DEK has been consistently associated with tumor progression and chemoresistance.s-10 Several lines of evidence, such as DEK-dependent formation of papilloma in a mouse model of skin cancerogenesis, 10 have led to its classification as a bona fide oncogene. In melanomas, high levels of DEK expression correlate positively with metastatic potential. chemoresistance and poor treatment outcome. Significantly, DEK expression levels can distinguish benign nevi from malignant melanoma, raising the possibility of using DEK as a tumor marker. • 9We and others have shown that DEK is involved in DNA repair and the response to DNA damage: cells with downregulated DEK expression are hypersensitive to genotoxic insults and show increased susceptibility to sublethal doses of DNA damaging agents. •12 DNA repair is affected by DEK ablation, as DNA strand breaks (DSBs) are repaired less efficiently and nonhomologous end-joining appears to be defective. 12 · 13 DEK is also a substrate for covalent and n...

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DNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

Depletion of calstabin1 (FKBP12) from the RyR1 channel and consequential calcium leakage from the sarcoplasmic reticulum (SR) is found in certain disease conditions such as dystrophy, aging or muscle overuse. Here, we first assessed the effect of calstabin1 depletion on resting Ca2+ levels and transients. We found that depletion of calstabin1 with the calstabin1-dissociation compound FK506 increased the release of calcium from the SR by 14 % during tetanic stimulation (50 Hz, 300 ms) and delayed cytosolic calcium removal. However, we did not find a significant increase in resting cytosolic Ca2+ levels. Therefore, we tested if increased SERCA activity could counterbalance calcium leakage. By measuring the energy utilization of muscle fibers with and without FK506 treatment, we observed that FK506-treatment increased oxygen consumption by 125% compared to baseline levels. Finally, we found that pretreatment of muscle fibers with the RyR1 stabilizer JTV-519 led to an almost complete normalization of calcium flux dynamics and energy utilization. We conclude that cytosolic calcium levels are mostly preserved in conditions with leaky RyR1 channels due to increased SERCA activity. Therefore, we suggest that RyR1 leakiness might lead to chronic metabolic stress, followed by cellular damage, and RyR1 stabilizers could potentially protect diseased muscle tissue.

24DNA replication stress is a major source of genomic instability and is closely linked to 25 tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) 26 enzymes are activated in response to replication stress resulting in poly(ADP-ribose) 27 (PAR) synthesis. PARylation plays an important role in the remodelling and repair of 28 impaired replication forks, providing a rationale for targeting highly replicative cancer 29 cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone 30 chromatin architectural protein whose deregulated expression is associated with the 31 development of a wide variety of human cancers. Recently, we showed that DEK is a 32 high-affinity target of PARylation and that it promotes the progression of impaired 33 replication forks. Here, we investigated a potential functional link between PAR and 34 DEK in the context of replication stress. Under conditions of mild replication stress 35 induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion 36 by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication 37 fork progression is dependent on DEK expression. Reducing DEK protein levels also 38 overcomes the restart impairment of stalled forks provoked by blocking PARylation. 39Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is 40 crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive 41 foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved 42 microscopy that DEK is not directly associated with the replisome since it binds to DNA 43 at the stage of chromatin formation. Our report sheds new light on the still enigmatic 44 molecular functions of DEK and suggests that DEK expression levels may influence 45 the sensitivity of cancer cells to PARP1/2 inhibitors. 46 3 47 48 Poly(ADP-ribosyl)ation (PARylation) is an abundant protein posttranslational 49 modification regulating numerous cellular functions among which the maintenance of 50 genomic stability plays a prominent role [1]. The enzyme responsible for 85-90% of 51 the cellular PAR synthesis activity is PARP1, with PARP2 accounting for the remainder 52 [2]. PAR can be covalently linked to and/or interact non-covalently with target proteins. 53 PARylation is highly dynamic and can be very transient in nature due to the activity of 54 the de-modifying enzyme, the PAR glycohydrolase or PARG [3]. Inhibition of 55 PARylation by small molecule compounds is a recently approved strategy for the 56 treatment of ovarian cancer [4]. The rationale for the use of PARP1/2 inhibitors in 57 chemotherapy is based on their synthetic lethal interaction with DNA damaging agents 58 in cells which are deficient for recombinational DNA repair through mutations in 59 BRCA1/2 [5, 6]. In these cells, inhibition of PARylation abrogates base excision repair 60 thereby turning endogenous single strand breaks (SSBs) in highly toxic, non-61 repairable double strand breaks (DSBs). In addition, P...

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