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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, Um Pot1 harbors an extra N-terminal OB-fold domain (OB-N), which was recently shown to be present in most metazoans. Um Pot1 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. 

Abstract Duplex telomere binding proteins exhibit considerable structural and functional diversity in fungi. Herein we interrogate the activities and functions of two Myb-containing, duplex telomere repeat-binding factors inUstilago maydis, a basidiomycete that is evolutionarily distant from the standard fungi. These two telomere-binding proteins,UmTay1 andUmTrf2, despite having distinct domain structures, exhibit comparable affinities and sequence specificity for the canonical telomere repeats.UmTay1 specializes in promoting telomere replication and an ALT-like pathway, most likely by modulating the helicase activity of Blm.UmTrf2, in contrast, is critical for telomere protection; transcriptional repression ofUmtrf2leads to severe growth defects and profound telomere aberrations. Comparative analysis ofUmTay1 homologs in different phyla reveals broad functional diversity for this protein family and provides a case study for how DNA-binding proteins can acquire and lose functions at various chromosomal locations. Our findings also point to stimulatory effect of telomere protein on ALT inUstilago maydisthat may be conserved in other systems.