Summary
A bioRxiv preprint integrates genome-wide maps of DNA breaks, replication and transcription in U2-OS and HeLa cancer cell lines. It reports that prolonged low-dose DNA polymerase inhibition redirects breaks toward late-replicating regions and identifies MUS81 as a regulator of recurrent break clusters.
A bioRxiv preprint presents an integrated genome-wide resource for studying where DNA double-strand breaks form in cancer cells and how replication stress changes those locations. The analysis combined maps of DNA breaks with measurements of DNA replication, replication timing, transcription and R-loops in two widely used human cancer cell lines: U2-OS and HeLa.
The researchers examined the cells under steady-state conditions and after prolonged exposure to a low dose of a DNA polymerase inhibitor. DNA polymerases copy DNA during replication; inhibiting them can slow or disrupt that process, creating replication stress. The study reports that the genomic environments associated with DNA breakage differed between the two conditions.
A genome-wide map of replication-associated damage
DNA double-strand breaks are lesions in which both strands of the DNA double helix are severed. If they are repaired inaccurately or persist during cell division, they can contribute to genome rearrangements and instability, a characteristic feature of cancer biology.
The resource assembled several types of genomic information to examine why breaks arise in particular regions. These included the positions of DNA breaks, sites where replication begins, the timing of replication across the genome, transcription activity and R-loops. R-loops are structures in which an RNA strand pairs with one DNA strand, leaving the other DNA strand exposed; they can form during transcription and affect genome maintenance.
Under steady-state conditions, endogenous DNA breaks were concentrated in origin-rich initiation zones. These are regions containing many replication origins, the points at which DNA copying can begin. The researchers also found enrichment of R-loops in these initiation zones, linking the structures with this particular replication environment.
Replication stress redirected breaks to late-replicating regions
Prolonged low-dose DNA polymerase inhibition changed the pattern. DNA break formation shifted toward origin-poor regions that replicate late in the cell cycle. Among the replication features examined, the study identified replication initiation zones and late-replicating regions as especially sensitive to prolonged replication stress.
The relationship between R-loops and genome fragility was not uniform. R-loops were enriched at replication initiation zones, but depleted from late-replicating regions and from recurrent DNA break clusters, or RDCs. This indicates that the contribution of R-loops depends on the surrounding replication environment rather than following one pattern across the genome.
The analysis also found that replication stress induced RDCs within long, actively transcribed genes. These clusters only partly overlapped with common fragile sites, genomic regions traditionally associated with breakage under replication stress. The result points to a broader and more context-dependent distribution of replication-associated damage than a map based only on common fragile sites would capture.
MUS81 helped control recurrent break clusters
Functional experiments identified the structure-specific endonuclease MUS81 as a major regulator of RDC formation. Removing MUS81 increased the number of recurrent DNA break clusters, while restoring its catalytic activity reduced them.
The authors interpret this pattern as evidence that MUS81 helps process or resolve replication intermediates before they persist into fragile, late-replicating regions. Replication intermediates are temporary DNA structures formed during copying; if they are not properly handled, they can become sources of chromosome breaks.
The study is a preprint rather than a peer-reviewed research article, and its experimental evidence comes from two cancer cell lines. The findings therefore provide a cell-based framework for understanding how replication architecture, transcription and DNA-processing activity shape genome fragility, while their relevance to human tumours or treatment responses requires further study.