Holloman, W. K. | bioRxiv Science

bioRxiv2021

Analysis of Ustilago maydis Pot1 reveals context-dependent, dichotomous regulation of homology-directed DNA repair factors by a telomere protein

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 latters strand exchange activity. Deleting the OB-N domain, which is implicated in Rad51-binding, caused telomere shortening, suggesting that Pot1-Rad51 interaction facilitates telomere replication. 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.

molecular biology↗

bioRxiv2020

Structurally distinct duplex telomere repeat-binding proteins in Ustilago maydis execute specialized, non-overlapping functions in telomere recombination and telomere protection

Duplex telomere binding proteins exhibit considerable structural and functional diversity in different phyla. Herein we address the distinct properties and functions of two Myb-containing, duplex telomere repeat-binding factors in Ustilago maydis, a basidiomycete fungus that is evolutionarily distant from the standard budding and fission yeasts. The two telomere-binding proteins in U. maydis, named UmTrf1 and UmTrf2, have different domain organizations and belong to distinct protein families with different phylogenetic distributions. Despite these differences, they exhibit comparable affinities and similar sequence specificity for the canonical, 6-base-pair telomere repeats. Deletion of trf1 triggers preferential loss of long telomere tracts, suggesting a role for the encoded protein in promoting telomere replication. Trf1 loss also partially suppresses the ALT-like phenotypes of ku70-deficient mutants, suggesting a novel role for a telomere protein in stimulating ALT-related pathways. In keeping with these ideas, we found that purified Trf1 can modulate the helicase activity of Blm, a conserved telomere replication and recombination factor. In contrast, trf2 appears to be essential and transcriptional repression of this gene leads to severe growth defects and profound telomere aberrations that encompass telomere length heterogeneity, accumulation of extrachromosomal telomere repeats such as C-circles, and high levels of single-stranded telomere DNA. These observations support a critical role for UmTrf2 in telomere protection. Together, our findings point to a unique, unprecedented division of labor between the two major duplex telomere repeat-binding factors in Ustilago maydis. Comparative analysis of UmTrf1 homologs in different phyla reveals a high degree of functional diversity for this protein family, and provides a case study for how a sequence-specific DNA binding protein can acquire and lose functions at different chromosomal locations.

molecular biology↗