Genetic and biochemical analysis of DNA damage checkpoint signalling in budding yeast

• Applicant: Professor Dr. Boris Pfander
• Subject Area: Cell Biology
• Term: from 2012 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 225776363

DNA is constantly being damaged by agents form intrinsic and extrinsic sources. This poses a threat to cellular survival and the stability of the genomic information and has do be dealt with by the cellular DNA damage response. In eukaryotes a conserved signalling pathway called the DNA damage checkpoint regulates many aspects of the cellular program in response to DNA lesions. The apical checkpoint machinery is responsible for the recognition of DNA lesions and the transmission of the checkpoint signal to downstream effectors. Checkpoint kinases are the principle signal transducing enzymes, and they are supported by a network of non-kinase mediators, which function as co-sensors, regulators and scaffolds. Apical checkpoint signalling takes place localized on chromatin near sites of DNA damage. While the importance of the co-localization of the signalling molecules has been established, the exact spatial organisation remains to be identified. In the first project we will determine the localisation, composition and stoichiometry of chromatin-associated checkpoint signalling complexes. Knowledge of this molecular layout will put us in a better position to resolve the network of apparently redundant functional interactions that constitutes the DNA damage checkpoint. First, we will investigate the DNA binding and localization of checkpoint proteins with regard to a site-specific DNA lesion and compare the nature and requirements of checkpoint protein assemblies close to a DNA damage site with those that have spread into the surrounding chromatin. Second, we aim to identify the molecular composition of these assemblies using a combination of cross-linking, affinity purification and quantitative proteomics and characterize these complexes with regard to stoichiometry and requirements for formation. The checkpoint kinases on top of the checkpoint signalling cascade are referred to as initiator or apical checkpoint kinases. The activation of these apical checkpoint kinases is regulated by activator proteins. Their regulation in turn will thus determine overall checkpoint activation. In a second project, we will investigate two possible ways of activator regulation: the recruitment and localization to sites of checkpoint signalling and the regulation of the activity by post-translational modifications. In budding yeast currently two activators are known and we will screen for further proteins that may share this ability. The functional characterization of new and known activators will aim to identify specificities for substrates, location and cell cycle stage.

• DFG Programme: Research Grants