Single-stranded telomeric repeats segregate into spatial compartments within ALT-associated PML bodies

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Abstract 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. Graphical abstract Highlights 3D MINFLUX combined with exchange DNA-PAINT resolves APB components at ∽3 nm isotropic precision, enabling direct visualization of molecular organization beyond the reach of conventional super-resolution microscopy. PML forms a partially open, ∼70 nm thick spherical shell in APBs with reduced molecular density compared to canonical PML nuclear bodies, providing the structural scaffold and coordinate system for mapping internal APB organization. Double-stranded telomeric chromatin (TRF1-marked) fills the APB interior, with additional TRF1 domains outside the shell indicative of multi-telomere clustering at individual APBs. ssC-rich repeats concentrate in the inner part of the PML shell while ssG-rich DNA repeats distribute broadly through the interior, revealing strand-specific spatial segregation. The ssG-rich single-stranded repeats in APBs predominantly comprise DNA and partially colocalize with POT1, connecting their spatial distribution to single-stranded telomeric DNA substrates. The spatial organization of APB components motivates a mechanistic model in which t-loop processing generates C-circle templates for rolling-circle amplification, while multi-telomere clustering facilitates inter-telomeric recombination.

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