Ustilago maydis telomere protein Pot1 harbors an extra N-terminal OB fold and regulates homology-directed DNA repair factors in a dichotomous and context-dependent manner

Abstract

The telomere G-strand binding protein Pot1 plays multifaceted roles in telomere maintenance and protection. We examined the structure and activities of Pot1 in Ustilago maydis, a fungal model that recapitulates key features of mammalian telomere regulation. Compared to the well-characterized primate and fission yeast Pot1 orthologs, UmPot1 harbors an extra N-terminal OB-fold domain (OB-N), which was recently shown to be present in most metazoans. UmPot1 binds directly to Rad51 and regulates the latter’s strand exchange activity. Deleting the OB-N domain, which is implicated in Rad51-binding, caused telomere shortening, suggesting that Pot1-Rad51 interaction facilitates telomere maintenance. Depleting Pot1 through transcriptional repression triggered growth arrest as well as rampant recombination, leading to multiple telomere aberrations. In addition, telomere repeat RNAs transcribed from both the G- and C-strand were dramatically up-regulated, and this was accompanied by elevated levels of telomere RNA-DNA hybrids. Telomere abnormalities of pot1-deficient cells were suppressed, and cell viability was restored by the deletion of genes encoding Rad51 or Brh2 (the BRCA2 ortholog), indicating that homology-directed repair (HDR) proteins are key mediators of telomere aberrations and cellular toxicity. Together, these observations underscore the complex physical and functional interactions between Pot1 and DNA repair factors, leading to context-dependent and dichotomous effects of HDR proteins on telomere maintenance and protection.

Comparative genomics ofCryptococcusandKwoniellareveals pathogenesis evolution and contrasting karyotype dynamics via intercentromeric recombination or chromosome fusion

“…Other mechanisms for chromosome fusion include dysfunction in telomere protection, as seen in human cells with compromised shelterin complexes leading to dicentric fusions [134–136]. While similar defects in shelterin subunits or telomerase in Kwoniella could be promoting chromosome fusions, our analysis of did not reveal obvious defects in shelterin subunits or telomerase compared with Ustilago maydis [83, 137, 138], although the RNA subunit of telomerase in Kwoniella remains unidentified. While challenging to detect bioinformatically, innovative approaches like those used in U. maydis [139, 140] could be key.…”

Ustilago maydisTrf2 ensures genome stability by antagonizing Blm-mediated telomere recombination: fine-tuning DNA repair factor activity at telomeres through opposing regulations

“…We found that similar to the pot1 mutant, in both Blm-overexpressing and Trf2-deficient cells, the telomere de-protection is accompanied by elevated levels of both G- and C-strand telomere repeat RNAs [33]. These findings suggest that increased transcription of both telomere strands is a general consequence of telomere de-protection.…”

“…Our recent analysis of Pot1-deficient cells indicates that upon telomere deprotection, both the G-and Cstrand of telomere repeats are transcribed at abnormally high levels, suggesting that deprotected telomeres are substrates for the transcription apparatus (Zahid et al, 2022). There is also growing evidence that DNAs flanking DSBs are actively transcribed, pointing to another resemblance between DSBs and deprotected telomeres (Ohle et al, 2016;Liu et al, 2021;Sharma et al, 2021).…”

Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity

“…The blots were hybridized to an oligonucleotide probe carrying 3 copies of the C. glabrata telomere repeat unit (CgTELCx3: [GCACCCAGACCCCACA] 3 ) and analyzed using a PhosphorImager scanner and ImageQuant software. Average telomere lengths for individual TRF clusters were estimated using a previously reported method ( 47 ).…”