DNA replication is a vulnerable process. Perturbations in DNA replication, which lead to slowing or collapse of replication forks, is defined as “replication stress”. It is now clear that endogenous replication stress is the main driver of genome instability in early cancer development, and inducing DNA replication stress is a commonly employed anti-cancer chemotherapeutic strategy. However, while it is clear that replication stress induces cell cycle arrest or cell death, the underlying mechanisms remain unclear.

Lethal replication stress has been previously associated with “mitotic catastrophe”, a broad descriptor encompassing the poorly understood and complex mechanisms that connect genomic insult to mitotic disruption and cell death. In addition, low dosages of replication stress drive genome instability through the passage of damaged DNA and chromosome segregation errors during mitosis. In this project, we used cell and molecular biology, assayed primarily by live- and fixed cell imaging, to explore the connectivity between genomic DNA replication stress in S-phase, with alterations in chromosome structure and cellular outcomes in mitosis.

In cells with a functional p53, we found replication stress induced a direct transition from G2 to G1-phase, i.e. “mitotic bypass”, and growth arrest. However, in p53-compromised cells, lethal replication stress induced mitotic cell death in the same cell cycle. Under these conditions, genomic DNA replication stress induced centromere cohesion errors in S/G2, mediated through activity of the cohesin antagonist WAPL. WAPL-dependent, aberrant chromosomal structures, were then passed from G2 to M, which activated and maintained the spindle assembly checkpoint (SAC) to propagate mitotic arrest. During mitotic arrest, WAPL further destabilized cohesion at the centromeres to drive cell death through the separation of sister chromatids by microtubule pulling forces: a phenomena known as “cohesion fatigue”. Also during mitotic arrest, an independent and parallel mechanism, regulated by the mitotic kinase Aurora B and the telomere protein TRF2, actively destabilized the terminal chromosome end structures. This induced a telomere-specific ATM-dependent DNA damage response as a secondary mechanism of cell death.

Cumulatively, our data define a mechanism of replication stress-dependent cell death through mitotic catastrophe. We conclude that in the absence of tumour suppressor pathways, mitosis functions as a critical clearing house, which recognizes and exacerbates chromosome structural aberrations, to drive cell death and maintain genome stability.