Abstract
Recent research has shown, that the biological processes of DNA replication, DNA damage, cell cycle and mitosis cannot be considered as isolated cellular functions but are mechanistically linked in many ways. For instance, when cells are exposed to replication stress and enter mitosis with unresolved replication intermediates, it can give rise to chromosome lesions, which are then transmitted to the next cell cycle. Aberrations in the mitotic process itself can potentially give rise to post-mitotic DNA damage with serious onsequences for genome integrity in the ensuing cell generations. The relative proportion and crosstalk between these causative versus consequent
genome-destabilizing events remains elusive. The aim of this thesis was to assess the relationship between DNA damage and mitotic perturbations. Using large-scale, real-time siRNA screens and a live cell imaging approach complemented by immunofluorescence (IF) analysis in fixed cells, we were able to correlate the impact of mitotic perturbations with the occurrence of DNA damage. Surprisingly, we saw that siRNA-mediated knockdown of only a subset of mitotic genes was accompanied by an increase in DNA damage, showing that even very dramatic chromosome segregation defects and massive aberrations in nuclear morphology and chromosomal ploidy can
happen without detectable DNA breakage. Despite the lack of DNA damage, almost all gene knockdown lead to an increased stress level detectable through an increase in p53 levels. We propose that this strong p53 response, which often occurs without detectable increase in DNA damage, is caused by the acute increase in chromosomal aneuploidy. Finally, our systematic approach to the DNA damage-mitosis crosstalk reveals widespread cell death in response to mitotic pertubations, showing that mitotic regulators are essential for cell viability and that their prolonged loss is not tolerated.
genome-destabilizing events remains elusive. The aim of this thesis was to assess the relationship between DNA damage and mitotic perturbations. Using large-scale, real-time siRNA screens and a live cell imaging approach complemented by immunofluorescence (IF) analysis in fixed cells, we were able to correlate the impact of mitotic perturbations with the occurrence of DNA damage. Surprisingly, we saw that siRNA-mediated knockdown of only a subset of mitotic genes was accompanied by an increase in DNA damage, showing that even very dramatic chromosome segregation defects and massive aberrations in nuclear morphology and chromosomal ploidy can
happen without detectable DNA breakage. Despite the lack of DNA damage, almost all gene knockdown lead to an increased stress level detectable through an increase in p53 levels. We propose that this strong p53 response, which often occurs without detectable increase in DNA damage, is caused by the acute increase in chromosomal aneuploidy. Finally, our systematic approach to the DNA damage-mitosis crosstalk reveals widespread cell death in response to mitotic pertubations, showing that mitotic regulators are essential for cell viability and that their prolonged loss is not tolerated.
| Original language | English |
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| Publisher | Faculty of Science, University of Copenhagen |
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| Number of pages | 143 |
| Publication status | Published - 2014 |