DNA Replication Stress Generates Distinctive Landscapes of DNA Copy Number Alterations and Chromosome Scale Losses

Background We previously showed that a major driver of cancer chromosomal instability (CIN) is replication stress, the slowing or stalling of DNA replication. However, the precise drivers of replication stress in cancer and the mechanisms by which these cause CIN and influence tumour evolution remain unclear. Common fragile sites are well-known genomic locations of breakage after aphidicolin-induced replication stress, but their precise causes of fragility are debated, and additional genomic consequences of replication stress are not fully explored.

Results Using single cell sequencing we detected DNA copy number alterations (CNAs) caused by one cell cycle under replication stress in diploid non-transformed cells. Aphidicolin-induced replication stress caused multiple types of CNAs associated with different genomic regions and features. Coupling cell type-specific analysis of CNAs to gene expression and single cell replication timing analyses allowed us to pinpoint the causative large genes of the most recurrent chromosome-scale CNAs. In RPE1 cells these were largely confined to three sites on chromosomes 1, 2 and 7 and generated acentric lagging chromatin and micronuclei containing these chromosomes. Different replicative stresses generated distinct profiles of CNAs providing the potential to interpret specific replication stress mechanisms from cancer cells.

Conclusions Chromosomal instability driven by replication stress occurs via focal CNAs and chromosome arm-scale changes, with the latter confined to a very small subset of chromosome regions, potentially heavily skewing cancer genome evolution trajectories. Single cell CNA analysis thus reveals new insights into the impact of replication stress on the genome and provides a platform to further dissect molecular mechanisms involved in the replication stress response and to gain insights into how replication stress fuels chromosomal instability in cancer.