Date Published: August 13, 2019
Publisher: Public Library of Science
Author(s): Magdalena Ganz, Christopher Vogel, Christina Czada, Vera Jörke, Eva Christina Gwosch, Rebecca Kleiner, Agnieszka Pierzynska-Mach, Francesca Cella Zanacchi, Alberto Diaspro, Ferdinand Kappes, Alexander Bürkle, Elisa Ferrando-May, Domenico Maiorano.
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-ribosyl)ation (PARylation) is an abundant protein posttranslational modification regulating numerous cellular functions among which the maintenance of genomic stability plays a prominent role . The enzyme responsible for 85–90% of the cellular PAR synthesis activity is PARP1, with PARP2 accounting for the remainder . PAR can be covalently linked to and/or interact non-covalently with target proteins. PARylation is highly dynamic and can be very transient in nature due to the activity of the de-modifying enzyme, the PAR glycohydrolase or PARG . Inhibition of PARylation by small molecule compounds is a recently approved strategy for the treatment of ovarian cancer . The rationale for the use of PARP1/2 inhibitors in chemotherapy is based on their synthetic lethal interaction with DNA damaging agents in cells which are deficient for recombinational DNA repair through mutations in BRCA1/2 [5, 6]. In these cells, inhibition of PARylation abrogates base excision repair thereby turning endogenous single strand breaks (SSBs) in highly toxic, non-repairable double strand breaks (DSBs). In addition, PARP1/2 inhibitors possess DNA trapping activity which causes DSBs on its own due to the collision of PARP-DNA complexes with the DNA replication and transcription machineries . Impaired DNA replication has recently come into the focus as a further source of DNA lesions which can become lethal to cells treated with PARP1/2 inhibitors. If not removed timely, replication blocks lead to fork collapse leaving behind single ended DNA strand breaks as well as SSBs which require PARylation for their prompt repair. PARP1/2 was also shown to be directly involved in replication fork stabilization and protection. Thus, PARP is required for the restart of collapsed forks after prolonged exposure to hydroxyurea (HU) , protects transiently stalled forks from premature and extensive resection  and regulates fork reversal induced e.g. by low doses of camptothecin (CPT). More precisely, PARylation prevents RecQ helicase from resolving regressed forks prematurely, thus avoiding fork run off across DNA lesions and DSB generation [10, 11]. Finally, PARP1/2 was shown to play an important role also during unperturbed DNA replication. Using pharmacological PARG inhibition to stabilize and detect basal PAR levels, the polymer was shown to be required for sensing and repairing a sub-set of unligated Okazaki fragments thus providing a back-up pathway for the completion of lagging strand DNA synthesis .
In this study we have explored a potential functional relationship between DEK and PARP1/2 in the context of DNA replication stress. For both proteins, there is consistent evidence for their involvement in the response to impaired DNA replication. Both DEK and PARP1/2 preferentially bind to unconventional non-B DNA structures like cruciform and G4 DNA [37, 61–63]. These structures are difficult to replicate and particularly abundant in heterochromatin. Both DEK and PARP1/2 are found enriched in chromatin of S-phase cells [64–67] and have been associated with the formation and maintenance of heterochromatin [40, 68]. DEK was shown to modulate the efficiency of DNA replication in vitro , and, more recently, we showed that normal DEK levels are necessary to sustain replication fork progression and to prevent fork rearrangements in cells undergoing replication stress . DEK is a target for covalent modification by and non-covalent interaction with PAR. Covalent PARylation was reported to occur at glutamic acid 136  and 207 , arginine 208  and, most recently, at serine 279 . Based on sequence alignment, DEK was further proposed to harbour three non-covalent PAR-binding domains , of which the central one (aa position 195–222) shows the strongest binding affinity and mediates about 50% of the PAR-binding activity of the protein in vitro (this study and ).