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TRF2 is an essential and conserved double-strand telomere binding protein that stabilizes chromosome ends by suppressing DNA damage response and aberrant DNA repair. Herein we investigated the mechanisms and functions of the Trf2 ortholog in the basidiomycete fungus Ustilago maydis, which manifests strong resemblances to metazoans with regards to the telomere and DNA repair machinery. We showed that UmTrf2 binds to Blm in vitro and inhibits Blm-mediated unwinding of telomeric DNA substrates. Consistent with a similar inhibitory activity in vivo, over-expression of Trf2 induces telomere shortening, just like deletion of blm, which is required for efficient telomere replication. While the loss of Trf2 engenders growth arrest and multiple telomere aberrations, these defects are fully suppressed by the concurrent deletion of blm or mre11 (but not other DNA repair factors). Over-expression of Blm alone triggers aberrant telomere recombination and the accumulation of aberrant telomere structures, which are blocked by concurrent Trf2 over-expression. Together, these findings highlight the suppression of Blm as a key protective mechanism of Trf2. Notably, U. maydis harbors another double-strand telomere-binding protein (Tay1), which promotes Blm activity to ensure efficient replication. We found that deletion of tay1 partially suppresses the telomere aberration of Trf2-depleted cells. Our results thus point to opposing regulation of Blm helicase by telomere proteins as a strategy for optimizing both telomere maintenance and protection. We also show that aberrant transcription of both telomere G- and C-strand is a recurrent phenotype of telomere mutants, underscoring another potential similarity between double strand breaks and de-protected telomeres. Author summary: The ends of linear chromosomes are protected from abnormal repair by a collection of telomere proteins. One protein that plays an especially prominent role is TRF2, which binds to double-stranded telomere repeats. In this study, we analyzed the mechanisms and functions of Trf2 in a yeast-like fungus named Ustilago maydis, which manifests a high degree of similarity to animal cells with respect to telomere regulation. We showed that Trf2 binds directly to a conserved DNA helicase called Blm and inhibits the ability of Blm to unwind telomeric DNA in a purified, cell-free reaction. We also used over-expression and depletion of either Trf2 or Blm or both to demonstrate an inhibitory effect of Trf2 on Blm function in vivo. For example, depletion of Trf2 triggers Blm-dependent telomere aberrations and cell death. Interestingly, another double-strand telomere binding protein named Tay1 was found to stimulate Blm activity to promote telomere replication. Together, our results indicate that U. maydis optimizes Blm function through opposing regulation of its activity via distinct telomere proteins. We also detected high levels of abnormal transcripts that correspond to both strands of telomeres in a variety of telomere mutants, suggesting that de-protected telomeres are permissive substrates for the transcription apparatus. [ABSTRACT FROM AUTHOR]

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. Author summary: The key function of telomeres is to stabilize chromosome ends against abnormal repair by forming a special nucleoprotein structure. One protein that plays a conserved and critical function at telomeres is Pot1, which binds to the single-stranded DNA at the very tips of chromosomes. In this study, we investigated the function of Pot1 in a yeast-like fungus called Ustilago maydis, which exhibits strong similarities to animal cells with respect to DNA repair and telomere regulation. We showed that U. maydis Pot1 interacts with two major DNA repair factors (Rad51 and Brh2) in a context-dependent manner. In normal cells, Pot1 binds directly to Rad51, and this interaction facilitate the maintenance of telomere DNA. However, when Pot1 is depleted and telomeres become dysfunctional, Rad51 and Brh2 become toxic to the cell by promoting abnormal recombination at telomeres. Therefore, Pot1 also negatively regulates the recombination activities of Rad51 and Brh2. These results highlight the complexity of Pot1 mechanisms and provide a compelling demonstration of the antagonistic relationship between telomere proteins and DNA repair factors. Notably, in comparison to previously characterized Pot1, U. maydis Pot1 has a different domain architecture, which is similar to RPA1, and which suggests an evolutionary kinship between these two protein families. [ABSTRACT FROM AUTHOR]