Alternative lengthening of telomeres (ALT) is a recombination process that maintains telomeres in the absence of telomerase and helps cancer cells to survive. yeast has been used as a robust model of ALT; however, the inability to determine the frequency and structure of ALT survivors hinders understanding of the ALT mechanism. Here, using population and molecular genetics approaches, we overcome these problems and demonstrate that contrary to the current view, both RAD51-dependent and RAD51-independent mechanisms are required for a unified ALT survivor pathway. This conclusion is based on the calculation of ALT frequencies, as well as on ultra-long sequencing of ALT products that revealed hybrid sequences containing features attributed to both recombination pathways. Sequencing of ALT intermediates demonstrates that recombination begins with RAD51-mediated strand invasion to form DNA substrates that are matured by a RAD51-independent ssDNA annealing pathway. A similar unified ALT pathway may operate in other organisms, including humans.
