Faithful genome replication requires exact control of efficiency, time and location of initiation of DNA replication at thousands of replication origins. Even mild de-regulation of the first step of initiation, origin licensing, has been linked to genomic instability and human diseases. However, little is known about how the deregulation of the second step of initiation, origin firing, is linked to replication stress and subsequent mitotic errors. Our previous investigations helped to establish a major regulation hub of origin firing, the Treslin-MTBP protein complex. The first aim of our project addresses how replication stress through decreased origin firing (under-firing) can lead to mitotic error. We will down-regulate origin firing specifically and gradually using auxin-degron tagging of MTBP, partial RNAi of Treslin/MTBP and pharmacological inhibition of DDK in chromosomally stable HCT116 and DLD-1 cells. Subsequently, we will characterise in-depth the resulting replication stress and mitotic dysfunction, and investigate the underlying cellular mechanisms. Collaboration within the FOR2800 will enable us to asses genomic instability upon under-firing by using NGS-based genome analysis. Mathematical modelling with Maik Kschischo (FOR2800, SP3) will help us understand the dynamics of genome replication upon under-firing. Our second aim will address how mitotic failure and aneuploidy affects the correct regulation of replication origin firing. In collaboration with Zuzana Storchova (FOR2800, SP8) we will investigate the consequences of chronic and acute changes of chromosome numbers and extended and shortened mitotic timing on replication initiation and origin firing. Finally, we will use proximity biotinylation/mass spectrometry to identify regulation pathways of the MTBP-Treslin regulation hub that are de-regulated in response to mitotic dysfunction.