Abstract
Alternative lengthening of telomeres (ALT) is a recombination-based pathway that enables cancer cells to maintain telomere length. ALT establishment remains poorly understood due to difficulties in identifying its molecular steps. Here, using Oxford Nanopore sequencing and computational modeling, we track the evolution of individual chromosome end structures during ALT establishment in yeast and delineate three molecular milestones. First, homologous recombination via break-induced replication (BIR) at telomeres and sub-telomeric regions delays senescence. Second, BIR interruption and microhomology-mediated recombination promote initial telomere extension and telomeric circle formation. Third, the final extension—critical for chromosome-end stabilization—employs a highly mutagenic replication mechanism to copy telomeric circles. Linking these newly defined ALT milestones is Mph1, the homolog of human FANCM, which plays important roles throughout ALT establishment by disrupting BIR synthesis and promoting template switching. Our findings support a model in which template switching during DNA repair synthesis drives the progression through the multiple steps involved in ALT establishment and progression, ultimately producing ALT survivors.