Koeleman, E. S.; Kaplan, C.; do Rego Barros Fernandes Lima, M. A.; Braun, D. M.; Knotz, C.; Rippe, K.

Alternative lengthening of telomeres (ALT)-associated PML bodies (APBs) concentrate single-stranded (ss) telomeric DNA and RNA species that are critical for recombination-based telomere maintenance. However, how these species are organized inside APBs has remained invisible at microscopic resolution. Here, we map the nanoscale topology of APB components using 3D MINFLUX super-resolution microscopy combined with multiplexed exchange DNA-PAINT labeling at [~]3 nm localization precision. We discover that ssC-rich and ssG-rich telomeric repeats occupy distinct spatial compartments within a partially open, ~70 nm thick PML protein shell: ssC-rich repeats concentrate at the inner shell surface, while ssG-rich repeats distribute broadly through the interior alongside TRF1-marked double-stranded telomeric chromatin. The ssG-rich signal is predominantly DNA and frequently colocalizes with POT1 assemblies. Additional TRF1 clusters outside the shell indicate multi-telomere association. Together, these structural constraints motivate a model of ALT in which t-loop resolution generates a C-circle template that drives rolling-circle amplification of telomeric repeats.