Consequences of unscheduled DNA replication

• Applicant: Professor Dr. Boris Pfander
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 445098914

DNA replication underlies exquisite control in order to generate an accurate copy of the genome throughout eukaryotes. This control ensures that the genome is replicated exactly once per cell cycle and counteracts the occurrence of unscheduled DNA replication. Unscheduled DNA replication leads to so-called replication stress, but the link between the two phenomena is poorly understood. Replication stress in general is known to be a key driver of genome instability in healthy organisms as well as during cancer development. However, replication stress can have several causes and we currently lack a molecular marker that would allow to discriminate how replication stress has occurred. In our previous work we have established several model systems to induce different amounts of unscheduled DNA replication in the G1 phase of the cell cycle of Saccharomyces cerevisiae. This has allowed us to characterize the mechanism of DNA replication outside of S phase as well as cellular factors which limit its occurrence. These systems have also enabled us to study how unscheduled DNA replication generates replication stress and genome instability. Molecular biology and genomics data suggest that unscheduled replication leads to over-replication, which causes DNA breakage through replication collisions. Strand-specific mapping of single-stranded DNA has been instrumental to gain these mechanistic insights and our preliminary data suggest that the ssDNA-signature may offer the possibility to differentiate between different forms of replication stress. In the continuation of this project, using single-cell, single-molecule and advanced genomics techniques we (i) aim to test, whether replication collisions and DNA breakage are the major mechanism of genome instability after unscheduled DNA replication or whether additional mechanisms exist. We will (ii) improve our methodology to measure the accumulation of ssDNA quantitatively, with nucleotide resolution and including information on the length of individual ssDNA stretches by introducing and testing the ssDNA-seq workflow. Finally, we will (iii) generate a catalogue of ssDNA signatures after induction of well-defined forms of replication stress in yeast and human cells and test whether ssDNA signatures allow us to detect the occurrence of unscheduled replication after a deregulated G1-S-transition. This work will therefore not only deepen our understanding of how unscheduled DNA replication and genome instability are linked, but also will also show more broadly whether ssDNA can be harnessed as discriminatory marker for replication stress phenomena.

• DFG Programme: Research Grants