Workpackage 2
WP2 Cell Biology of DNA damage response and repair mechanisms
(partners involved: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13)
Cellular studies have revealed many aspects of DNA repair defects in mammals. Cell lines have been indispensable tools to clone DNA repair genes and to pinpoint the consequences of repair defects at the cellular level. Cells derived from patients have been particularly useful in these studies. However, a disadvantage of studies based on this material is the heterogeneous genetic composition of patient cells, which can obscure more subtle consequences of defects. This work package will therefore concentrate on work with cells derived from genetically homogeneous mouse models (generated in WP3). Subsequently, these data will then be compared to the human situation (WP5).
The collaborating labs have ample experience in isolating and growing several cell types derived from (genetically modified) mice. From many mouse models, mouse embryonic fibroblasts (MEFs) and/or mouse dermal fibroblasts (MDFs) have been generated, which can be used in these studies. In addition, we have embryonic stem (ES) cells from several mouse models, as well as keratinocytes. This collection will be extended if necessary. Further a complete set of yeast mutants altered in replication, recombination, repair and checkpoint pathways are available. We also have a solid expertise in RNAi-mediated gene knock-down to selectively deprive isogenic mouse (or human) derived cells from one or a combination of genome surveillance factors, providing the possibility to complement the above approaches.
These various cell types will be used to assess the role of genome care-takers with regard to their sensitivity to genotoxic agents, recovery of DNA and RNA synthesis after genotoxic treatment, mutagenesis, chromosomal aberrations, repair capacity, cell cycle progression and (apoptotic) cell death. Experimental approaches will include live cell studies (using relevant DNA repair proteins coupled to the Green Fluorescent Protein (GFP)) and chromatin immuno-precipitation (ChIP) assays. The outcome of this analysis will help to identify overlapping and specialised roles of surveillance pathways and possible interactions with other cellular pathways (e.g. transcription, replication, recombination).
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