Workpackage 1
WP1 Biochemistry and Structural Biology of DNA damage response and repair mechanisms
(partners involved: 1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 14)
Detailed knowledge of the biochemistry of protein-DNA interactions has been indispensable for most of our current knowledge about vital cellular processes such as transcription, replication and genome maintenance. The collaborating participants have studied the biochemistry and structural biology of the various DNA repair pathways. To obtain structural and mechanistic insights into various DNA surveillance processes we will use the collaborative expertise to reconstitute surveillance pathways in vitro, in order to mechanistically understand the underlying biochemistry. Nucleotide excision repair (NER) and base excision repair (BER) can now be reconstituted in vitro from purified proteins, which enables direct investigation of the effects of specific mutations in genes encoding the participating repair factors on the ability to carry out these repair reactions. The non-homologous end joining (NHEJ) reaction is functional in a cell-free system, but as yet this reaction has not been reconstituted from purified components. The biochemistry of MMR has been studied in great detail in E. coli, but, despite the fact that the repair process functions extremely efficiently in cell extracts, the reconstitution of mammalian MMR from purified proteins has not yet been achieved, as some of the components remain to be identified. Similarly, translesion synthesis (TLS) has been studied in much more detail in the bacterial system than in mammals, although it is becoming increasingly clear that this genome care-taking system is of great importance for mammals. The large number of proteins involved in homologous recombination (HR) suggests that this reaction will also be difficult to reconstitute in vitro. However, in all the areas of DNA repair research listed, the participating laboratories have gained new insights by studying the activities of separate components of these repair pathways that are relevant to their function in vivo. The established assays (e.g. mismatch recognition in MMR, strand invasion in HR or lesion bypass synthesis by TLS polymerases) can be used to directly investigate the effect of specific mutations on the activities of the proteins involved and may also serve as a starting point to assemble complete repair reactions from separate components. We will also investigate how DNA repair and checkpoint pathways are influenced by chromatin structure and are controlled during the cell cycle. It will also be important to investigate the structure and function of critical proteins acting in the signal transduction cascade (checkpoint) need to work this definition in earlier activated by DNA damage and to define the physical interactions of checkpoint factors with proteins involved in the different repair reactions. Finally, the attempts to reconstitute of these reactions with purified (recombinant) defined components may uncover the need for additional factors, which will then be isolated and characterized through all work packages.
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