Transcription at an inducible common fragile site reveals replication origin strength hierarchy.

While genome-wide analyses in vertebrates suggest that transcription negatively regulates replication origin activation, thereby shaping the landscape of replication initiation, it remains unknown whether efficient replication origins can overcome this suppression at common fragile sites (CFSs). In this study, we addressed this question by inserting two model origins into the avian DMD CFS and a transcriptionally silent late-replicating region in DT40 cells. The DMD gene, which is neither transcribed nor fragile in wild-type cells, became fragile following transcriptional activation in a genetically engineered cell line. Previous molecular combing experiments have shown that transcription represses origin firing. Here, we demonstrate that a minimal origin remains partially active under basal transcription conditions, yet is fully inactivated upon transcriptional induction. In contrast, the efficient β-actin promoter/origin retains functionality despite transcriptional activation. The same functional hierarchy emerges in a late-replicating, transcriptionally silent, and non-fragile locus. These results define a class of robust origins that function across diverse chromosomal contexts and support the model in which CFSs arise from transcription-dependent repression of replication origin initiation across large, late-replicating genes. Our study also suggests that depletion of cis-elements specifying efficient replication origins contributes to the formation of late-replicating domains, whereas a high density establishes early-replicating regions.