Workpackage 4
WP4 Systems Biology of DNA damage response and repair mechanisms. Genomics, proteomics and bioinformatics
(partners involved: 1, 2, 8, 9, 11, 12, 14)
We will analyse genome-wide transcriptome and proteome responses to DNA damage using micro-array, two dimensional-difference gel electrophoresis (2D-DIGE) and mass spec. The studies can produce surrogate markers to monitor the efficacy of DNA repair inhibitors in vivo (WP4), provide information regarding the state of these pathways in clinical material (WP5), and increase our understanding of cellular response to DNA damage. An additional benefit may be the identification of potential new drug targets within these DNA repair pathways. Participant 15 has the skill and expertise to assess the potential therapeutic value of these new targets and develop medium to high throughput assays to identify novel inhibitors.
Experimental design will be critical for obtaining interpretable data from both gene expression profiling and proteomic experiments. One has to keep in mind that the differentiation status of the cell, the exact cell type, genetic background and growth status all contribute to the gene expression profile and the proteomic content of the cell. We will therefore initially concentrate on defined cells from identical genetic backgrounds (except for one specific mutation) under identical growth conditions. Gene expression and proteomic profiles of cells with different mutations in DNA repair genes will be compared in unchallenged, as well as challenged cells. The types of challenging treatment will differ for the various repair mutants. NER mutants, for example, will be treated with a chemical that produces lesions that can only be repaired by NER (BPDE), or by UV irradiation, whereas cells deficient in the recently discovered AlkB homologous hABH2 and hABH3 will be treated with alkylating agents that preferentially introduce the type of alkylations recognized by these enzymes.
The gene expression profiling experiments in particular will produce many very large, data sets, which will require extensive bioinformatics facilities. Such facilities will be provided by participants 7 (Prof. Jiricny is chair of the steering committee of the Functional Genomics Centre Zurich), 1, 3, 6, and 11 (which have direct access to local- genomics and bioinformatics facilities). They will also work on developing new software to refine interpretation of these large data sets. We will give special attention to collection of the data sets to annotated databases and directed literature searches. In order to facilitate implementation of these techniques for the other participants, as well as comparison of data between the various labs, we will develop an interface to produce a uniform output for all participants. The data will be made available on a communal web site, which will be accessible for all participants. When appropriate, these data will also become open for the whole research community. This way of distributing data will not only be advantageous for the participants, but also for other researchers in Europe and the rest of the world. We will provide a platform for genomics and bioinformatics that will disseminate its know-how throughout the European research community.
The bioinformatics component of this work package will not only deal with the genomics part of this project, but also with analysis and modelling of protein dynamics in living cells (WP2). We have ample experience in this area, in which we developed techniques to monitor dynamic behaviour of GFP-tagged proteins in living cells and to model this behaviour in computer simulations. These analyses revealed that many multi-protein complexes involved in genome surveillance are not static, but instead are dynamic assemblies. They also showed that certain DNA repair proteins could become transiently immobilised upon induction of DNA damage. In several cases, computer simulations have proven to be indispensable to explain the data. We plan to extend our simulations as necessary.
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