Replication stress conferred by POT1 dysfunction promotes telomere relocalization to the nuclear pore

Abstract

Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we define the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.

The Sound of Silence: How Silenced Chromatin Orchestrates the Repair of Double-Strand Breaks

“…The dependency on the nuclear periphery for repair of heterochromatic DSBs appears analogous to the repair of persistent DSBs or collapsed replication forks in budding yeast, which also require the SUMOylation pathway to coordinate movement to the nuclear periphery [ 94 ]. Although the movement of heterochromatic DSBs to the nuclear periphery has not been described in mammalian cells to date, two recent studies have revealed that stressed-replication foci and dysfunctional telomeres can associate with the nuclear periphery in human cells [ 95 , 96 ]. Moreover, nuclear pore stability was found to be important to prevent telomere fragility, indicating the essentiality of the nuclear periphery in the maintenance of repetitive telomeric regions in humans [ 95 ].…”

Cancer‐associated POT1 mutations lead to telomere elongation without induction of a DNA damage response

“…In addition, caPOT1 mutations have been studied in human cells either by overexpression of caPOT1 alleles combined with and without the simultaneous depletion of wild-type POT1 in cancer cells or by engineering the homozygous mutations in cancer cell lines (Pinzaru et al, 2016;Chen et al, 2017;Gu et al, 2017;McMaster et al, 2018;Pinzaru et al, 2020). Under these conditions, POT1 mutations lead to telomere elongation and telomere deprotection.…”

Chromatin mobility and relocation in DNA repair

“…In human cells, cancer-associated mutant alleles of the telomere protein POT1 promote telomere-specific replication stress [60]. Complementary whole genome CRISPR interference and proximity-ligation proteomic screens revealed that replication stressed telomeres in mutant POT1 expressing cells associated with subunits of the NPC Y complex, nuclear basket, and NUP62 subcomplex [61]. Preventing POT1 mutant telomeres from associating with NPCs altered the type of DNA repair reactions that occurred at chromosome ends, consistent with specific DNA repair mechanisms ongoing at the nuclear periphery [61].…”

“…Examination of replication stressed human telomeres in mutant-POT1 cells revealed that inhibiting actin-dependent telomere relocalization to NPCs increased telomere fragility [61], which is a marker of telomere replication stress [55]. Further, preventing telomere-NUP interactions elevated telomere sister chromatid exchange (T-SCE) [61], signifying an increase in conventional telomere HDR events [72].…”