DNA topoisomerases participate in fragility of the oncogene RET

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Common fragile sites (CFS) are prone to DNA breakage during replication stress, often involving cancer-related genes like the RET oncogene.
• While DNA repair mechanisms at CFS are studied, the initial events causing breakage remain unclear.
• RET oncogene rearrangements are observed in thyroid cancer, suggesting a link to fragile site instability.

Purpose of the Study:

• To investigate the initial molecular events leading to aphidicolin (APH)-induced DNA breakage at common fragile sites.
• To map nucleotide-level breakpoints within the RET oncogene's fragile site in human thyroid cells.
• To determine the role of DNA topoisomerases in initiating fragile site breakage.

Main Methods:

• Mapped 144 aphidicolin (APH)-induced DNA breakpoints within RET intron 11 in human thyroid cells.
• Analyzed breakpoint locations relative to predicted DNA topoisomerase I/II cleavage sites and DNA secondary structures.
• Co-treated cells with APH and topoisomerase catalytic inhibitors (betulinic acid, merbarone) to assess effects on breakage.

Main Results:

• APH-induced breakpoints in RET intron 11 clustered at or near topoisomerase I/II predicted cleavage sites.
• Breakpoints also localized to DNA secondary structures recognized by topoisomerases.
• Inhibiting topoisomerase activity significantly reduced APH-induced breakage in RET intron 11 and FRA3B.

Conclusions:

• DNA topoisomerases I and II play a crucial role in initiating aphidicolin-induced common fragile site breakage.
• Topoisomerases may recognize and cleave DNA secondary structures formed during replication stress.
• These findings provide insight into the mechanisms underlying genomic instability at fragile sites in cancer.