The oncoprotein DEK affects the outcome of PARP1/2 inhibition during mild replication stress.

Magdalena Ganz,Agnieszka Pierzynska-Mach,Elisa Ferrando-May,Francesca Cella Zanacchi,Vera Jörke,Eva Christina Gwosch,Rebecca Kleiner,Alberto Diaspro,Christopher Vogel,Christina Czada,Alexander Bürkle,Ferdinand Kappes

doi:10.1371/journal.pone.0213130

Abstract

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.

Highlights

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 [1]
To investigate whether PARylation regulates the impact of DEK on the replication stress response, we set out from our previous observation that downregulation of DEK expression aggravates replication fork slowing induced by low concentrations of CPT
We examined the effect of DEK and PARP1/2 activity on CPT-induced replication fork progression using DNA fiber assays (Fig 1)

Summary

Introduction

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 [1]. 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). 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 providing a back-up pathway for the completion of lagging strand DNA synthesis [12]

Methods

Results

Conclusion