| Popis: |
Accurate replication of the genome during cell division is essential for maintaining genome integrity and suppressing diseases such as cancer. DNA replication forks can stall, collapse and/or break when they encounter obstacles present in DNA. This causes DNA replication stress, which is a major source of genome instability and mutations during carcinogenesis and cancer evolution. A number of studies have investigated how cells respond to replication stress by using various genotoxic agents such as radiation or therapeutic agents. However, these agents cause genome-wide damage; thereby, preventing control over where replication forks collapse or break, and preventing replication stress-associated molecular events from being studied directly. Here, we aimed to overcome these current methodological limitations by developing an innovative FLP-FRT system. The FLP-FRT system is designed to induce the generation of broken DNA replication forks at specific loci in mammalian cells. The system utilises a mutant FLP recombinase that binds to an integrated FLP recognition target site (FRT) marked with a florescent locus labelling system and generates an irreversible protein adduct and DNA single-strand break. Upon DNA replication, the fork encounters the gap leading to fork breakage and the formation of a single-ended DNA double-strand break. Using the ANCHOR labelling tool, the ANCH3-ANCH4 sites flanking the FRT sequence were simultaneously visualized. Our findings suggest that cells with a broken fork display a notable reduction in cell viability when compared to a direct DSB, suggesting they are more cytotoxic, as hypothesized. In conclusion, our study shows that the features of FRT-FLP system offers a widely applicable and powerful tool to directly study replication stress at a very high resolution at the damage site within single cells in vivo. This novel tool permits genetic, biological and microscopic analyses of broken replication fork resolution in mammalian cells, which can shed light on important aspects of cancer cell biology and the development of new therapeutic clinical strategies. |
