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.

Poly(ADP ribos)ylation (PARylation) is an impor tant posttranslational protein modification, and is involved in major cellular processes such as gene regulation and DNA repair. Its dysregulation has been linked to several diseases, including cancer. Despite its importance, methods to observe PARylation dynamics within cells are rare. By following a chemical biology approach, we developed a fluorescent NAD + analogue that proved to be a competitive building block for protein PARylation in vitro and in cells. This allowed us to directly monitor the turnover of PAR in living cells at DNA damage sites after near infrared (NIR) microirradiation. Addi tionally, covalent and noncovalent interactions of selected target proteins with PAR chains were visualized in cells by using FLIM FRET microscopy. Our results open up new opportunities for the study of protein PARylation in real time and in live cells, and will thus contribute to a better under standing of its significance in a cellular context.

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.

Poly(ADP-Ribos)ylierung (PARylierung) ist eine wichtige posttranslationale Proteinmodifikation,d ie in grundlegende zelluläre Prozesse wie Genregulation und DNA-Reparatur involviert ist. Ihre Fehlregulierung wurde mit verschiedenen Krankheiten wie Krebs in Verbindung gebracht. Trotz grçßter Wichtigkeit gibt es nur wenige Methoden, um PARylierung und ihre Dynamik in Zellen zu beobachten. Mittels einer chemisch-biologischen Herangehensweise entwickelten wir ein fluoreszierendes NAD + -Analogon, das sich als kompetitiver Baustein für die PARylierung von Proteinen in vitro und in Zellen erwies.D ies ermçglichte uns,d en Umsatz von PARd irekt und in lebenden Zellen nachD NA-Schädigung durchN IR-Mikrobestrahlung zu verfolgen. Zusätzlich wurden mithilfe von FLIM-FRET-Mikroskopie kovalente und nichtkovalente Interaktionen von PARm it ausgewählten Proteinen sichtbar gemacht. Unsere Ergebnisse erçffnen neue Chancen für die schnelle zelluläre Untersuchung der Protein-PARylierung in Echtzeit und werden somit zu einem besseren Verständnis und hçherer Aussagekraft im zellulären Kontext beitragen. Abbildung 1. Struktur von A) Poly(ADP-ribose) und B) NAD + und NAD + -Analoga 1 und 2,die in dieser Studie verwendet wurden.[ + + ]D iese Autoren haben zu gleichen Teilen zu der Arbeit beigetragen.Hintergrundinformationen zu diesem Beitrag sind unter: http://dx.

The correct inheritance of chromatin structure is key for maintaining genome function and cell identity and preventing cellular transformation. DEK, a conserved non-histone chromatin protein, has recognized tumor-promoting properties, its overexpression being associated with poor prognosis in various cancer types. At the cellular level, DEK displays pleiotropic functions, influencing differentiation, apoptosis, and stemness, but a characteristic oncogenic mechanism has remained elusive. Here we report the identification of DEK bodies, focal assemblies of DEK regularly occurring at specific, yet unidentified sites of heterochromatin replication exclusively in late S-phase. In these bodies, DEK localizes in direct proximity to active replisomes in agreement with a function in the early maturation of heterochromatin. A high-throughput siRNA screen, supported by mutational and biochemical analyses, identifies SUMO as a major regulator of DEK body formation, linking DEK to the complex SUMO protein network that controls chromatin states and cell fate. This work combines and refines our previous data on DEK as a factor essential for heterochromatin integrity and facilitating replication under stress and delineates an avenue of further study for unraveling DEK's contribution to cancer development.