Mutations are double edged sword. On one hand, most new mutations can be harmful. On the other hand, they’re the building blocks of adaptation and evolution, helping organisms survive and thrive over time. Major source of mutations are DNA replication errors despite multiple error avoidance and repair control mechanisms. That’s why it’s crucial to figure out how and where these errors appear in the genomes.

To tackle this, we harnessed powerful evolutionary conserved natural error correction machinery, mismatch repair system. We modulated this machinery in the way that it can detect but do not repair errors. By tagging mismatch repair protein MutL, with fluorescent proteins, we were able to visualize DNA replication errors as they emerge in living cells. By using MutL-ChIP-seq analysis we were able to identify replication error prone genomic region, along with associated genomic features and proteins.

Our findings reveal that replication error hotspots are non-randomly distributed along the chromosome and are enriched in sequences with distinct features: lower thermal stability facilitating DNA strand separation, mononucleotide repeats prone to DNA polymerase slippage, and sequences prone to forming secondary structures like cruciform structures, which increase likelihood of DNA polymerase stalling. These hotspots showed enrichment for binding sites of nucleoid-associated proteins and have high transcriptional activity.

In conclusion, our study provides a comprehensive genome-wide map of replication error hotspots, offering a holistic perspective on the intricate interplay between various mechanisms that can compromise the faithful transmission of genetic information.