In the course of this thesis, DEK was shown to be removed from chromatin in response to DSB induction. An increase in DEK protein mobility was measured shortly after damage infliction by multiphoton laser irradiation. The time needed to induce a suitable amount of DNA damage in a cube-shaped region of interest within the nucleus is dependent on the available laser power. Comparable levels of DNA damage can be achieved using either high or low laser power by adjusting pixel dwell time. The setup used during this thesis provided a laser power that required an irradiation time of 2 minutes for DNA damage
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induction. Accelerated mobility of DEK protein was detected directly after the irradiation procedure was completed. Ten minutes after damage induction, DEK mobility had decreased again to a level comparable to that of undamaged cells. Since the increase of DEK mobility occurs very rapidly after DSB induction, an improvement of the technique that would allow for shorter irradiation times would provide means to investigate DEK mobility immediately after damage induction. It can be predicted that mobility changes at earlier time points will be more pronounced. The immediate effect of laser microirradiation on DEK’s mobility is most likely a consequence of chromatin decondensation which is promoted by PARP1/2. DEK removal from chromatin has already been described in the context of chromatin decondensation during gene transcription, where it is mediated by SET and PARP (Gamble & Fisher, 2007). A similar mechanism might also be responsible for DEK’s removal from chromatin as a consequence of DNA damage induction. It is also possible that DEK removal is at least in part mediated by phosphorylation, but this point has still to be demonstrated. Generally, it would be a most valuable improvement to be able to study the mobility of endogenous DEK protein. The gene targeting methods using zinc-finger or TAL-effector nucleases as mentioned above also allow for endogenous gene tagging: a cDNA encoding GFP flanked by DNA sequences complementary to stretches up- and downstream of the endonuclease target site enables integration into the genome via endogenous HR repair. Tagging of endogenous DEK could provide the means to study DEK mobility and localization with respect to endogenous post-translational modification patterns in more detail.
Apart from this early response of DEK mobility to DSB induction, DEK was also shown to be removed from chromatin as a late and persistent consequence after DNA replication stress. Very small amounts of DEK residing in the cytoplasm but not in the nucleus could still be detected by immunocytochemistry. Re-localization of DEK to the cytoplasm was so far only reported in the context of active DEK secretion from monocyte-derived macrophages either as its free form or compartmentalized in exosomes (Mor-Vaknin et al, 2006). DEK secretion could be prevented by inhibition of CK2 which is the primary kinase responsible for DEK phosphorylation (Kappes et al, 2004). It is an open question, whether inhibition of CK2 also prevents DEK removal from the nucleus during recovery from DNA replication stress. However, DNA replication stress also induces sumoylation of proteins at the replication fork, such as PCNA (Moldovan et al, 2007), the RPA complex (Dou et al, 2010), and BLM (Ouyang et al, 2009). Protein modification by attachment of the ubiquitin-like protein SUMO is known to impact more on protein structure, protein-protein interaction, and protein-protein localization rather than on protein-protein stability. Since DEK contains one consensus sumoylation motif as well as additional non-consensus sites and was recently found to be sumoylated upon induction of oxidative stress (Grant, 2010), sumoylation of DEK was considered a possible cause for the change of DEK localization during recovery from DNA replication stress. Western blot analysis of immunoprecipitated
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DEK provided a first hint towards a modification of DEK with SUMO1. However, this modification still needs to be verified via complementary approaches, such as mass spectrometry analysis. Apart from the interesting question about the posttranslational modification responsible for the changes in DEK’s localization, the long persistence of the observed cytoplasmic localization is puzzling. During the recovery from HU-induced replication stress, DEK was virtually absent from cell nuclei for several days albeit cells were proliferating and had lost synchronicity again. Although it has to be formally proven, complete removal of DEK from chromatin will most likely result in global changes of chromatin structure. These changes can be considered as a long-term epigenetic memory of the experienced DNA replication stress. Recently, it was shown that DNA replication stress affects chromatin assembly and epigenetic memory by impacting on histone recycling (Jasencakova et al, 2010). To my knowledge a long-term alteration of global chromatin architecture as a response to DNA replication stress is a complete new finding.
The results on DEK localization presented in this thesis provide the basis for further study of DEK as an interesting candidate involved in epigenetic memory of DNA replication stress.
Conclusion
Taken together, this thesis uncovered an important role of DEK in the cellular response to DNA replication stress, particularly in replication fork progression and processing of replication stress-induced DNA damage. It provides a link between DEK’s known DNA repair facilitating activity on the one hand and its tumor promoting properties on the other hand. By promoting the stabilization of arrested replication forks, possibly mediated via FANCD2, DEK counteracts the accumulation of ATR substrates under DNA replication stress. By doing so, DEK aids to circumvent the replication stress-induced DNA damage barrier against cancerogenesis. Therefore, this thesis provides a fundamental explanation for how DEK’s positive effect on DNA repair benefits cell proliferation and tumor development.
7 MATERIAL & METHODS
If not stated otherwise, all chemicals were purchased from Sigma Aldrich and fluorescently labeled secondary antibodies from Invitrogen. Cell culture reagents were from Gibco, Invitrogen. All used cell culture material was from Corning. Cell culture dishes for live cell imaging and imaging of mitotic cells were from ibidi.