DEK counteracts DNA damage arising from perturbed DNA replication

DNA replication problems as occurring e.g. at atypical DNA structures can culminate in incompletely replicated DNA and the formation of DNA strand breaks (Debatisse et al, 2012; Dillon et al, 2010; Ma et al, 2012). Having observed that DEK is required for efficient fork progression when replication is impaired, it was determined whether DEK also impacts on the handling of DNA lesions induced by replication stress. To this end, the formation of nuclear DNA repair foci resulting from the recruitment of marker proteins to sites of DNA damage was compared in control and shDEK cells treated with replication inhibitors. Foci were visualized via indirect immunocytochemistry. S-phase cells were identified via labeling with cyclin A (CYCA)-specific antibodies or EdU incorporation. To avoid bias, confocal images of cell nuclei were processed automatically using a custom-designed ImageJ macro. First, the formation of 53BP1 and γH2AX foci after exposure of cells to low doses of HU (Figure 5.8) and APH (Figure 5.9 and 5.10) was investigated.

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Figure 5.8 | The DNA damage response to HU-induced replication stress is influenced by DEK expression.

(A) Confocal images of U2OS control (top row) and shDEK cells (bottom row) treated with HU (1 mM) for 2 h (right panel) or left untreated (left panel). Cells were labeled with 53BP1 and CYCA-specific antibodies.

Scale bar: 5 µm. (B+C) Quantification of HU-induced DNA-damage in U2OS (B) and HeLaS3 (C) control and shDEK cells treated as in (A). Confocal images of at least 100 CYCA-positive cells were analyzed per

sample. Graph shows the mean of three independent experiments. Error bars represent SEM. T-test:

* p≤ 0.05.

Replication inhibition via HU very effectively induced 53BP1-positive foci in S phase cells. Treatment with HU yielded a marked increase in 53BP1-positive foci in shDEK cells as compared to controls in U2OS and HeLa S3 cells (Figure 5.8). In shDEK cells treated with APH, a slightly increased number of γH2AX positive S-phase cells was observed as compared to control (Figure 5.9 A+B), whereas no 53BP1 foci were induced by APH in these cells (data not shown). Phosphorylation of H2AX as specific marker for DNA double strand break formation has been recently disputed, since this histone modification is observed also after UV irradiation and exposure to high salt (Baure et al, 2009; Cleaver et al, 2011; de Feraudy et al, 2010; Marti et al, 2006; Revet et al, 2011). These agents both lead to distortions of the DNA and chromatin structure but not directly to its breaking. A more reliable indicator of DNA strand breaks, in particular of those occurring during replication, is the concomitant occurrence of 53BP1 and γH2AX-specific signals at nuclear foci (de Feraudy et al, 2010; Ray Chaudhuri et al, 2012). Since foci positive for both markers were not observed in APH-treated S-phase cells, it can be concluded that low doses of this agent do not primarily cause DNA strand breaks in the employed cellular system but rather lead to the formation of unusual DNA intermediates as a consequence of impeded replication, which in turn activate a DNA damage response.

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Figure 5.9 | The DNA damage response to APH-induced replication stress is influenced by DEK expression.

(A) Confocal immunofluorescence images of U2OS control (top row) and shDEK (bottom row) cells treated with 200 nM APH for 24 h (left panel) or left untreated (right panel). Cells were then labeled with EdU (10 µM) for 30 min and γH2AX and Rad51 were detected with specific antibodies. EdU was visualized by labeling with Alexa-488-Azide. (B, C, D) Analysis of confocal immunofluorescence images of at least 100 EdU-positve cells per sample treated as in (A). The mean foci number per cell with SEM of three independent experiments is displayed. T-test: * p≤ 0.05.

The effect of DEK on the response to replication stress-induced DNA damage was further corroborated by analysis of the formation of RAD51 and FANCD2 foci. RAD51 has functions both in DNA strand break repair via homologous recombination and in DNA replication where it protects the nascent DNA strand from MRE11-mediated cleavage (Hashimoto et al, 2010). FANCD2, a protein of the Fanconi Anemia pathway, recruits to stalled replication forks and is monoubiquitinated in a γH2AX-dependent manner (Bogliolo et al, 2007). There, FANCD2 also contributes to maintain the integrity of single stranded regions (Renaud & Rosselli, 2013; Schlacher et al, 2012).

In control cells, the overall number of RAD51 positive foci was slightly decreased upon APH treatment (Figure 5.9 C), whereas double positive foci for RAD51 and γH2AX were marginally elevated (Figure 5.9 D). In contrast, APH treatment led to a significant increase of the total number of RAD51 positive foci (Figure 5.9 C) as well as RAD51/γH2AX co-localization in shDEK cells (Figure 5.9 D). Surprisingly, the opposite effect was observed when investigating the formation of FANCD2 positive foci upon APH treatment (Figure 5.10): FANCD2 foci were induced massively in DEK expressing control S phase cells whereas shDEK cells displayed only a moderate increase in FANCD2 positive foci (Figure 5.10 A+C). The same tendencies were also evident during mitosis. The APH concentration had to be decreased by a factor of four to obtain sufficient number of cells in mitosis.

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FANCD2 foci were also induced on anaphase chromosomes and foci numbers in control cells were slightly elevated compared to shDEK cells.

Figure 5.10 | DEK promotes formation of FANCD2-dependent repair of replication stress-induced DNA damage. (A) Confocal images of U2OS control and shDEK cells treated with APH (200 nM) for 24 h or left untreated. FANCD2 and CYCA were detected via specific antibodies. Scale bar: 5 µm. (B) Maximum intensity-projections of epifluorescence z-stacks of U2OS control and shDEK cells treated with APH (50 nM). FANCD2 was detected using a specific antibody. (C) Quantitative analysis of cells treated as in A (S phase) and B (mitosis). FANCD2 foci were counted in CYCA-positive S phase cells (at least 100 cells were evaluated per sample) and on anaphase chromosomes from three experiments (untr: 25 cells, APH:

47 cells). Graph shows mean number of FANCD2-positive foci per anaphase cell.

Since repetitive elements in the genome are very likely prone to hinder replication fork progression, and chromosome centromeres typically contain such repetitive DNA elements, it was investigated whether APH induced DNA damage in centromeric regions or induced fragile sites outside of centromers. Co-staining of FANCD2 and centromeres using the CREST antibody revealed that DNA damage induction did occur predominantly outside of centromeric regions and most likely was related to the expression of common fragile sites (Figure 5.10 A).

The reciprocal response of shDEK cells and control cells with respect to RAD51 and FANCD2 foci formation suggests that DEK might play a role in fork protection or in the repair of collapsed forks by supporting FANCD2 function. Taken together, these results provide evidence that DEK protects from DNA lesions arising from replication stress and that it most likely exerts this function by influencing the processing of DNA damage at stalled replication forks.

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5.2.5 DEK protects daughter cell generations from replication