Replication stress can drive genetic instability and is associated with chromosomal instability in cancer and during reprogramming of stem cells, however the exact mechanisms connecting replication stress to chromosome aberrations are not known. Here we use single cell DNA sequencing to precisely map DNA copy number alterations (CNAs) after one cell division cycle under replication stress in two diploid cell types to megabase pair resolution. Unexpectedly, we find that replication stress generates several distinct classes of CNAs, and that specific genomic regions convert to particular classes of CNA. By combining analyses of single cell replication timing and bulk gene expression from both cell types, we provide a comprehensive picture of the features and mechanisms converting replication stress to DNA copy number alterations. The breakpoints of large CNAs tend to be associated with late DNA replication timing, low surrounding gene expression and proximity to large or giant genes. By contrast, small amplifications show contrasting associations and may represent a new class of replication stress induced CNAs. These findings provide a platform to further dissect molecular mechanisms involved in the replication stress response and to gain insights into how replication stress can drive chromosomal instability in cancer.

Replication Stress Generates Multiple Distinct Classes of Copy Number Alterations