DEK’s effect on cell proliferation and replication fork progression

It is a well-established concept that oncogene-induced DNA replication stress is an important source of DNA damage arising during cancer development. This DNA damage, if accumulating, can activate the above mentioned barrier against tumor development resulting in senescent or apoptotic cells. Such cells are permanently removed from the actively proliferating cell population. Proliferation pressure in transformed cells selects for survivors that are able to circumvent this barrier. DEK was already shown to disable this barrier by modulating p53 stability leading to apoptosis inhibition. The induction of differentiation and senescence were also demonstrated to be inhibited or delayed when DEK expression is elevated. The fact that DEK protects from DNA damage and promotes its repair had not been considered as potential mechanisms to circumvent this barrier. To test this hypothesis, the impact of DEK on DNA damage induction and DNA replication itself was investigated in this thesis in the context of different replication stress scenarios.

DEK downregulation impaired proliferation, in particular when cells were challenged by depletion of deoxynucleotides or by mild polymerase inhibition. The G2/M arrest observed via flow cytometry analysis of control and DEK-depleted cells is in accordance with a previous report where untreated HeLa cells displayed a slight G2/M arrest upon DEK

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downregulation alone (Kappes et al, 2011). However, it cannot be ruled out that the overall cell cycle distribution under replication stress can be additionally affected by a G1 arrest that delays cell entry into the subsequent S phase as long as the stress situation still persists.

The negative effect of DEK downregulation on cell cycle progression and proliferation, combined with previous reports on a link between DEK and DNA replication motivated the investigation of DEK’s involvement in replication fork progression, in particular when cells experience replication stress. DNA fiber analysis revealed that DEK depletion results in a decrease in replication efficiency in untreated cells. Replication stress further enhanced this effect. Interestingly, inhibition of PARP1/2 activity led to a comparable decrease in replication efficiency in DEK expressing control cells but had no additional effect on shDEK cells. The observation that PARP1/2 inhibition further decelerated fork progression is in contrast to the data from Ray Chaudhuri et al. (2012), who observed that PARP1/2 inhibition by Olaparib prevents - rather than enhances - fork slowing induced by CPT-mediated replication stress. This discrepancy might be explained by the different time window used for PARP1/2 inhibition: Ray Chaudhuri and colleagues pre-treated cells with Olaparib for five hours before pulse labeling and the inhibitor was present throughout both CldU- and IdU-incubation steps. In the experiments presented in this thesis, treatment with the PARP1/2 inhibitor Olaparib was limited to the duration of the second nucleotide pulse (IdU, 20 min) to avoid secondary effects of long-term PARP inhibition. Whether PARP1/2 inhibition for several hours is appropriate for investigating the involvement of PARylation in the acute handling of replication stress is a matter of debate: PARP1/2 activity was demonstrated to affect not only DNA repair mechanisms but also to modulate overall chromatin structure by PARylation of histones and non-histone chromatin proteins (reviewed in detail in: (Beneke, 2012; Faraone-Mennella, 2005; Kraus & Lis, 2003;

Quenet et al, 2009)), to impact on gene transcription by co-regulation of transcription factors (extensively reviewed in: (Kraus, 2008; Kraus & Hottiger, 2013; Kraus & Lis, 2003)), and to modulate DNA methylation by regulating the activity of DNA methyltranserase DNMT1 (Reale et al, 2005) again leading to alterations in gene expression. In this thesis, interference with PARP1/2 activity aimed at studying PARP1/2 involvement at impeded replication forks was kept as short as possible to minimize the impact on gene regulation. Olaparib is highly membrane permeable, and inhibits PARP activity within 5 minutes after its addition to the cell culture medium allowing effective short-term PARP1/2 inhibition. The effect of Olaparib was verified by inhibition of PAR-dependent XRCC1 accumulation at DSBs in living cells (data not shown; and Martin Stöckl, unpublished data).

The fact that PARP1/2 inhibition barely affected replication fork speed in CPT-treated shDEK cells whereas in control cells there was a significant additional retardation as

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compared to CPT treatment alone points to a functional link of DEK and PARP1/2 at the replication fork. These findings further suggest that either covalent PARylation of DEK or non-covalent interaction of DEK and PAR is necessary for DEK’s protective activity at the replication fork under replication stress conditions. To clarify this interesting aspect it is necessary to analyze DEK’s modification in more detail, especially in the context of replication stress. The dissection of target sites for PARylation or the identification of PAR interacting sites that are responsible for DEK’s activity at the replication fork would deepen the current understanding of the joint function of DEK and PARP at the replication fork.

Furthermore, it is still an open question whether the observed DEK-mediated facilitation of replication fork progression also translates into a faster completion of genome duplication in control cells compared to shDEK cells. It was recently demonstrated in Chinese hamster cells that replication fork velocity affects origin firing (Courbet et al, 2008). The authors reported that a reduction of replication fork speed resulted in the activation of latent origins enabling the timely completion of genome duplication. In addition, defects in homologous recombination were shown to affect the spatial proximity of activated origins: inactivation of either RAD51, XRCC2 or BRCA2 in hamster cell lines resulted in a reduced replication fork speed, which was compensated by an increased firing of replication origins (Daboussi et al, 2008). Further studies on DNA replication with respect to DEK expression should address the question whether DEK only affects replication fork velocity or whether it also impacts on origin firing. DNA fiber spreads as performed in the course of this thesis is not the suitable technique to adress this question. However, DNA fiber stretching is a valuable method that allows conclusions about origin firing. This technique should be employed in the near future for a more detailed study of DEK’s action during DNA replication.

Additionally, it has to be considered that shRNA-mediated downregulation of DEK expression never leads to a complete ablation of the protein. Thus, residual DEK might still exert its function during replication. Genomic knockout of DEK most likely will lead to more severe and unequivocal phenotypes. Gene knockout is achievable in cultured cells via novel gene targeting methods, using for example zinc-finger nucleases (Urnov et al, 2010) or TALENs (Cermak et al, 2011) designed to introduce DNA double strand breaks at a specific site in the genome. The repair of these breaks employs endogenous DNA repair mechanisms such as homologous recombination (HR) and non-homologous end joining (NHEJ), the latter being accompanied by the integration of a random number of nucleotides at the damaged site, eventually leading to frame shifts that result in complete loss of expression of the target gene. A completely DEK deficient human cell model would be a most valuable improvement for the study of DEK function.

If and how DEK influences DNA topology at or near the replication fork is not clear, but DEK’s ability to introduce positive supercoils in vitro (Waldmann et al, 2002) might

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provide the means to fine tune supercoiling near the fork in a way that is beneficial for replication fork stability and progression. Whether DEK induces positive supercoiling at or near the replication fork in living cells remains to be demonstrated. In vitro data on the effect of supercoiling on DNA replication showed that increased supercoiling ahead of the replication fork induced fork regression (McGlynn & Lloyd, 2001). In addition, DNA stretches and DSBs thereby facilitating cell cycle progression. This interpretation also accommodates the observation that DEK was not only important for fork integrity and speed but also resulted in lower numbers of γH2AX-positive DNA damage foci upon replication stress induction by APH. Beside DEK’s ability to change DNA topology, its histone chaperone activity (Kappes et al, 2011; Sawatsubashi et al, 2010) could also positively impinge on replication fork progression. During replication, histones of the parental DNA strand are recycled and used for chromatin assembly of the daughter strands.

The shuttling of histones from the parental to the newly synthesized strands is mediated by histone chaperones and the efficiency of histone re-location directly affects replication fork velocity (Abe et al, 2011; Groth et al, 2007; Nabatiyan & Krude, 2004). Thus, histone supply at the replication fork might be negatively affected by DEK downregulation leading to a decelerated fork progression. Replication stress can lead to a trapping of histones by histone chaperones which – after removal of the fork blocking lesion – can be integrated into newly synthesized DNA strands as soon as replication proceeds (Jasencakova et al, 2010). Therefore, DEK downregulation might affect histone supply at the fork during normal progression and during recovery from replication stress.

6.2.2 DEK’s impact on DNA replication stress susceptibility and