Abstract
Alternative lengthening of telomeres (ALT) is a recombination-based pathway enabling cancer cells to maintain telomeres. ALT establishment remains poorly understood due to difficulties 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—utilizes 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 where template switching during DNA repair synthesis drives the transitioning through the multiple steps involved in ALT establishment and progression, ultimately producing ALT survivors.
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Requests for unique/stable reagents generated in this study and further information and resources should be directed to the corresponding author. All ONT sequencing generated in this study have been deposited in NCBI’s BioProject with the accession code PRJNA1254968. Source data are provided with this paper.
Code availability
The code used to analyze the telomere sequencing data is available at GitHub (https://github.com/Jacob-M-Wells/TeloTracker)90. The code for the computational modeling of telomere dynamics is available at GitHub (https://github.com/josep-comeron/Yeast-Telomere-Dynamics)91.
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Acknowledgements
We thank Dr. James E. Haber and the members of the Malkova Lab for insightful discussions and comments on this manuscript. This work was supported by NIA grant R01AG081263 to A.M. and J.M.C, by NIGMS grant R35GM127006 to AM, and by RR240030 grant from CPRIT to A.M.
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M-C.T., J.M.W., Anna Malkova, and J.M.C. designed the study. M-C.T., K.A.R., and K.M. performed genetic and molecular biological experiments. J.M.W., J.M.C., and R.P. developed bioinformatic pipelines. J.M.W., A.Malkov, and J.M.C. performed bioinformatic analysis. J.M.C. performed computational modeling. M-C.T., J.M.W., K.A.R., K.M., Anna Malkova, and J.M.C. analyzed the data. M-C.T., J.M.W., K.A.R., Anna Malkova, and J.M.C. wrote the manuscript.
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Tsai, MC., Wells, J.M., Renninger, K.A. et al. Highly mutagenic copying of telomeric circles promotes ALT establishment. Nat Commun (2026). https://doi.org/10.1038/s41467-026-72032-4
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DOI: https://doi.org/10.1038/s41467-026-72032-4
