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.

We present a three-color femtosecond Er/Yb:fiber laser enabling highly specific and standardized nonlinear optical manipulation of live cells. The system simultaneously provides bandwidth-limited 80-fs pulses with identical intensity envelope centered at wavelengths of 515, 775, and 1035 nm in the focus of a confocal microscope. We achieve this goal by combining high-order dispersion control via, for example, chirped fiber Bragg gratings with proper bandwidth management in each nonlinear conversion step. Wavelength-selective and noninterfering induction of deoxyribonucleic acid (DNA) photoproducts and DNA strand breaks, as well as fluorescence photoactivation of a photoactivatable green fluorescent protein (PA-GFP)-histone fusion protein, are demonstrated. The capability to introduce different types of DNA lesions and perform photoswitching experiments in a selective manner is essential for quantitative studies on DNA repair and chromatin dynamics.

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.

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...