Initiation and timely completion of DNA replication is an essential cellular function that must occur with high-fidelity, whist simultaneously navigating the structures imposed by the complex chromatin environment. Topological associated domains (TADs) are chromatin structures that are intimately linked with replication initiation and replication timing. Whether TADs influence replication initiation and the subsequent dynamics of DNA polymerization during replication stress is unclear.
To address these unknowns, we intersected features of chromatin structure (Hi-C) and replication timing (Repli-seq) with replication initiation and dynamics data (Okazaki fragment sequencing and nanopore long read sequencing) in RPE1-hTERT cells challenged with hydroxyurea to induce mild replication stress. We found replication initiation is enriched at TAD boundaries in early S-phase, during both unperturbed replication and replication stress. During mild replication stress there is an increase in the number of initiation zones and increased strength in replication fork directionality, suggesting forks are moving longer genomic distances.
We then evaluated the effect of replication dynamics on S-phase transcription using scRNAseq. We first identified S-phase cells within the data then conducted differential gene expression analysis. Overwhelmingly, we found genes were down regulated during replication stress, and these genes were enriched in replication initiation zones within TAD boundaries in early S-phase.
Together our data are consistent with TADs being important during replication stress, where new replication is initiated from TAD boundaries to maintain DNA polymerisation. We speculate the decrease in gene expression at TAD boundaries in early S-phase removes an obstacle for the replication machinery, accounting for the increased strength in replication fork directionality. As the passage of replication through chromatin is highly disruptive to its structure, understanding the functional role of chromatin during replication is essential to ensure the underlying DNA is not vulnerable to damage during this process.