MMEJ repair of breaks at TA repeats maintains ecDNA and cancer fitness.

Extrachromosomal DNA (ecDNA) comprises megabase-sized circular DNA elements that frequently carry oncogene amplifications, driving aggressive tumour phenotypes, therapeutic resistance and poor clinical outcomes across many cancers1-4. Although ecDNA is thought to arise from canonical double-strand break repair, the pathways that maintain it remain unclear. Here we show that inhibition of microhomology-mediated end joining, but not non-homologous end joining or homologous recombination, selectively depletes ecDNA, induces ecDNA-specific damage and promotes its sequestration into micronuclei, compromising the fitness of cancer cells that depend on ecDNA-driven oncogene amplification. Mechanistically, TA-rich loci on ecDNA are hotspots for DNA damage and breakage5,6. The DNA translocase FANCM suppresses break formation at these sites, while breaks that escape FANCM surveillance are cleaved by ERCC1-ERCC4 and channelled into microhomology-mediated end joining for repair. Single-cell whole-genome sequencing shows that disrupting FANCM or polymerase θ (Polθ) in COLO320DM cells causes structural instability characterized by deletions and small duplications, with breakpoints enriched at TA-rich regions. This fragility is recapitulated in human tumours, in which ecDNA rearrangements are enriched at TA repeats. Collectively, our findings reveal TA repeat fragility as an intrinsic vulnerability of circular DNA and identify Polθ inhibition as a promising strategy to potentially destabilize ecDNA and sensitize ecDNA-driven tumours to therapeutic intervention.