Locus-specific transcription silencing rescues replication stress-induced CNV and CFS formation at late replicating large genes. Homo sapiens x Mus musculus hybrid cell line isolate:GM11713

Partial inhibition of DNA replication leads to de novo copy number variant (CNV) formation throughout the genome, with accumulations at specific genomic loci that coincide with common chromosome fragile sites (CFSs). Here, we directly compared aphidicolin (APH)-induced CNV and CFS frequency between wild-type (WT) cells and isogenic cells in which FHIT gene transcription was ablated by CRISPR-Cas9-induced promoter deletion. APH treatment led to critical replication delays that remained unresolved in G2/M in the body of many, but not all, large transcribed genes, an effect that was specifically reversed at FHIT by deletion of its promotor. In contrast, neither over-expression nor knockdown of RNase H1 changed CNV or CFS induction frequency at large genes, suggesting that R-loops are not the primary mediator of the transcription effect. These results provide direct evidence in a controlled isogenic cell system that the transcription of large CFS genes is a determining factor for their replication stress-induced instability, and supports models in which instability mainly results from the transcription-dependent passage of unreplicated DNA into mitosis due to impaired replication recovery