Replication origin firing regulation to ensure genome stability in metazoan cells

• Applicant: Professor Dr. Dominik Boos
• Subject Area: Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 448973587

We propose to investigate the regulation of replication origin firing, the generation of replication forks, in metazoan cells. Origin firing regulation in normal and DNA damage conditions is critical for faithful genome duplication in all dividing cells and, thus, for avoiding genetic diseases and cancer. In metazoans, the main regulator proteins of origin firing were identified only recently and have therefore been little investigated. In normal conditions, enough origins must fire with appropriate timing and location to ensure that genome duplication is complete. In DNA damage conditions, origin firing is inhibited to avoid genetic alterations through replicating damaged templates. We and others described a regulation platform of origin firing, the MTBP-Treslin-TopBP1 protein complex (Boos et al. 2011, 2013). We identified MTBP as an origin firing factor (Boos et al. 2013) and recently presented a detailed characterisation of MTBP (Köhler et al. 2019): MTBP fulfils conserved roles through its N- and C-terminal domains that are conserved with the yeast core origin firing factor Sld7. MTBP also has metazoa-specific roles through its metazoa-specific middle domain. One such function of the middle domain involves the binding to the transcription-regulating Cdk8-cyclin C kinase (CDK8 kinase) that had not previously been implicated in replication. In this project we will:Aim A) Investigate the cellular roles and the molecular mechanisms of MTBP cooperation with the CDK8 kinase to regulate origin firing.Our preliminary data suggest that CDK8 has a direct role in establishing the metazoan genome replication program independently of its classic function in transcription. We will characterise the exact CDK8 role in cellular replication, identify and investigate potential CDK8 substrates, and clarify molecular mechanisms. Aim B) Investigate a new pathway of origin firing suppression via inhibitory phosphorylation of MTBP. Our preliminary data suggest that this MTBP phosphorylation may play a role in suppressing origin firing upon DNA damage. Surprisingly, we also found evidence for a role in genome replication in unperturbed cells. We will investigate in detail the underlying cellular and molecular mechanisms.Our approach will be to manipulate MTBP, CDK8 and other proteins in cultured human cells using RNAi, specific protein mutants and chemical inhibition to characterise replication functions. Replication will be analysed in detail using 1) standard methods to measure overall cellular replication efficiency and 2) several specific techniques to determine in detail the dynamics, timing and structure of cellular replication and origin firing. Recombinant proteins and mass spectrometry will serve to identify kinase substrate sites.Our project promises to advance our molecular understanding how metazoan cells regulate origin firing in normal and DNA damage conditions.

• DFG Programme: Research Grants