Transcription Represses Origin Activity in a Late-Replicating Fragile Site

Genome-wide analyses in vertebrates suggest that transcription can negatively regulate replication origin activation, thereby shaping the global landscape of replication initiation. In this study, we investigated how transcription and late replication influence replication origin activity by inserting an efficient minimal model origin into the avian DMD common fragile site (CFS) and a second transcriptionally silent late-replicating region. The DMD gene, which is neither transcribed nor fragile in wild-type DT40 cells, became fragile following transcriptional activation in a genetically engineered DT40 cell line. Previous molecular combing experiments have shown that transcription represses origin firing within the 500 kb 5’ half of the gene. Here, we demonstrate that when a minimal origin is inserted into this repressive region, it remains partially active under basal transcription conditions but is fully inactivated upon transcriptional induction. In contrast, inserting the efficient β-actin promoter/origin at the same site retains origin functionality despite transcriptional activation. Introducing these two origins into a late-replicating, transcriptionally silent locus reveals the same functional hierarchy. Notably, this contrasts with their comparable activity in a mid-late replicating region. These results define a class of replication origins capable of maintaining functionality across diverse chromosomal contexts. Our findings support the model in which CFSs arise due to transcription-dependent repression of replication origin initiation across large, late-replicating genes. Moreover, our results reinforce the idea that replication initiation in late-replicating genomic compartments is intrinsically less efficient than earlier-replicating regions.