Eun Young Yu

SLX4 Assembles a Telomere Maintenance Toolkit by Bridging Multiple Endonucleases with Telomeres

Summary SLX4 interacts with several endonucleases to resolve structural barriers in DNA metabolism. SLX4 also interacts with telomeric protein TRF2 in human cells. The molecular mechanism of these interactions at telomeres remains unknown. Here, we report the crystal structure of the TRF2-binding motif of SLX4 (SLX4TBM) in complex with the TRFH domain of TRF2 (TRF2TRFH) and map the interactions of SLX4 with endonucleases SLX1, XPF, and MUS81. TRF2 recognizes a unique HxLxP motif on SLX4 via the peptide-binding site in its TRFH domain. Telomeric localization of SLX4 and associated nucleases depend on the SLX4-endonuclease and SLX4-TRF2 interactions and the protein levels of SLX4 and TRF2. SLX4 assembles an endonuclease toolkit that negatively regulates telomere length via SLX1-catalyzed nucleolytic resolution of telomere DNA structures. We propose that the SLX4-TRF2 complex serves as a double-layer scaffold bridging multiple endonucleases with telomeres for recombination-based telomere maintenance.

A proposed OB-fold with a protein-interaction surface in Candida albicans telomerase protein Est3.

Ever shorter telomeres 3 (Est3) is an essential telomerase regulatory subunit thought to be unique to budding yeasts. Here we use multiple sequence alignment and hidden Markov model–hidden Markov model (HMM-HMM) comparison to uncover potential similarities between Est3 and the mammalian telomeric protein Tpp1. Analysis of site-specific mutants of Candida albicans Est3 revealed functional distinctions between residues that are conserved between Est3 and Tpp1 and those that are unique to Est3. Although both types of residues are important for telomere maintenance in vivo, only the former contributes to telomerase activity in vitro and facilitates the association of Est3 with telomerase core components. Consistent with a function in protein-protein interaction, the residues common to Est3 and Tpp1 map to one face of an OB-fold model structure, away from the canonical nucleic acid binding surface. We propose that Est3 and the OB-fold domain of Tpp1 mediate a conserved function in telomerase regulation.

Regulation of Telomere Structure and Functions by Subunits of the INO80 Chromatin Remodeling Complex

ABSTRACT ATP-dependent chromatin remodeling complexes have been implicated in the regulation of transcription, replication, and more recently DNA double-strand break repair. Here we report that the Ies3p subunit of the Saccharomyces cerevisiae INO80 chromatin remodeling complex interacts with a conserved tetratricopeptide repeat domain of the telomerase protein Est1p. Deletion of IES3 and some other subunits of the complex induced telomere elongation and altered telomere position effect. In telomerase-negative mutants, loss of Ies3p delayed the emergence of recombinational survivors and stimulated the formation of extrachromosomal telomeric circles in survivors. Deletion of IES3 also resulted in heightened levels of telomere-telomere fusions in telomerase-deficient strains. In addition, a delay in survivor formation was observed in an Arp8p-deficient mutant. Because Arp8p is required for the chromatin remodeling activity of the INO80 complex, the complex may promote recombinational telomere maintenance by altering chromatin structure. Consistent with this notion, we observed preferential localization of multiple subunits of the INO80 complex to telomeres. Our results reveal novel functions for a subunit of the telomerase complex and the INO80 chromatin remodeling complex.

Mutual Dependence of Candida albicans Est1p and Est3p in Telomerase Assembly and Activation

ABSTRACT Telomerase is an RNA-protein complex responsible for extending one strand of the telomere terminal repeats. Analysis of the telomerase complex in budding yeasts has revealed the presence of one catalytic protein subunit (Est2p/TERT) and at least two noncatalytic components (Est1p and Est3p). The TERT subunit is essential for telomerase catalysis, while the functions of Est1p and Est3p have not been precisely elucidated. In an earlier study, we showed that telomerase derived from a Candida est1-null mutant is defective in primer utilization in vitro; it exhibits reduced initiation and processivity on primers that terminate in two regions of the telomere repeat. Here we show that telomerase derived from a Candida est3-null mutant has nearly identical defects in primer utilization and processivity. Further analysis revealed an unexpected mutual dependence of Est1p and Est3p in their assembly into the full telomerase complex, which accounts for the similarity between the mutant enzymes. We also developed an affinity isolation and an in vitro reconstitution protocol for the telomerase complex that will facilitate future mechanistic studies.

Rap1 in Candida albicans: an Unusual Structural Organization and a Critical Function in Suppressing Telomere Recombination

ABSTRACT Rap1 (repressor activator protein 1) is a conserved multifunctional protein initially identified as a transcriptional regulator of ribosomal protein genes in Saccharomyces cerevisiae but subsequently shown to play diverse functions at multiple chromosomal loci, including telomeres. The function of Rap1 appears to be evolutionarily plastic, especially in the budding yeast lineages. We report here our biochemical and molecular genetic characterizations of Candida albicans Rap1, which exhibits an unusual, miniaturized domain organization in comparison to the S. cerevisiae homologue. We show that in contrast to S. cerevisiae, C. albicans RAP1 is not essential for cell viability but is critical for maintaining normal telomere length and structure. The rap1 null mutant exhibits drastic telomere-length dysregulation and accumulates high levels of telomere circles, which can be largely attributed to aberrant recombination activities at telomeres. Analysis of combination mutants indicates that Rap1 and other telomere proteins mediate overlapping but nonredundant roles in telomere protection. Consistent with the telomere phenotypes of the mutant, C. albicans Rap1 is localized to telomeres in vivo and recognizes the unusual telomere repeat unit with high affinity and sequence specificity in vitro. The DNA-binding Myb domain of C. albicans Rap1 is sufficient to suppress most of the telomere aberrations observed in the null mutant. Notably, we were unable to detect specific binding of C. albicans Rap1 to gene promoters in vivo or in vitro, suggesting that its functions are more circumscribed in this organism. Our findings provide insights on the evolution and mechanistic plasticity of a widely conserved and functionally critical telomere component.

Analyses of Candida Cdc13 Orthologues Revealed a Novel OB Fold Dimer Arrangement, Dimerization-Assisted DNA Binding, and Substantial Structural Differences between Cdc13 and RPA70.

ABSTRACT The budding yeast Cdc13-Stn1-Ten1 complex is crucial for telomere protection and has been proposed to resemble the RPA complex structurally and functionally. The Cdc13 homologues in Candida species are unusually small and lack two conserved domains previously implicated in telomere regulation, thus raising interesting questions concerning the mechanisms and evolution of these proteins. In this report, we show that the unusually small Cdc13 homologue in Candida albicans is indeed a regulator of telomere lengths and that it associates with telomere DNA in vivo. We demonstrated high-affinity telomere DNA binding by C. tropicalis Cdc13 (CtCdc13) and found that dimerization of this protein through its OB4 domain is important for high-affinity DNA binding. Interestingly, CtCdc13-DNA complex formation appears to involve primarily recognition of multiple copies of a six-nucleotide element (GGATGT) that is shared by many Candida telomere repeats. We also determined the crystal structure of the OB4 domain of C. glabrata Cdc13, which revealed a novel mechanism of OB fold dimerization. The structure also exhibits marked differences to the C-terminal OB fold of RPA70, thus arguing against a close evolutionary kinship between these two proteins. Our findings provide new insights on the mechanisms and evolution of a critical telomere end binding protein.

Telomere recombination pathways: tales of several unhappy marriages

All happy families are alike; each unhappy family is unhappy in its own way.–Leo Tolstoy, Anna Karenina

A popular engagement at the ends

Three recent studies converged on a specific protein-protein interface between TPP1 and telomerase as being crucial for the regulation of both telomerase recruitment and processivity in mammalian cells. An equivalent interaction appears to exist in budding yeast, making this a nearly universal means of telomerase regulation.

Brh2 and Rad51 promote telomere maintenance in Ustilago maydis, a new model system of DNA repair proteins at telomeres.

Recent studies implicate a number of DNA repair proteins in mammalian telomere maintenance. However, because several key repair proteins in mammals are missing from the well-studied budding and fission yeast, their roles at telomeres cannot be modeled in standard fungi. In this report, we explored the dimorphic fungus Ustilago maydis as an alternative model for telomere research. This fungus, which belongs to the phylum Basidiomycota, has a telomere repeat unit that is identical to the mammalian repeat, as well as a constellation of DNA repair proteins that more closely mimic the mammalian collection. We showed that the two core components of homology-directed repair (HDR) in U. maydis, namely Brh2 and Rad51, both promote telomere maintenance in telomerase positive cells, just like in mammals. In addition, we found that Brh2 is localized to telomeres in vivo, suggesting that it acts directly at chromosome ends. We surveyed a series of mutants with DNA repair defects, and found many of them to have short telomeres. Our results indicate that factors involved in DNA repair are probably also needed for optimal telomere maintenance in U. maydis, and that this fungus is a useful alternative model system for telomere research.

Fungal Ku prevents permanent cell cycle arrest by suppressing DNA damage signaling at telomeres

The Ku heterodimer serves in the initial step in repairing DNA double-strand breaks by the non-homologous end-joining pathway. Besides this key function, Ku also plays a role in other cellular processes including telomere maintenance. Inactivation of Ku can lead to DNA repair defects and telomere aberrations. In model organisms where Ku has been studied, inactivation can lead to DNA repair defects and telomere aberrations. In general Ku deficient mutants are viable, but a notable exception to this is human where Ku has been found to be essential. Here we report that similar to the situation in human Ku is required for cell proliferation in the fungus Ustilago maydis. Using conditional strains for Ku expression, we found that cells arrest permanently in G2 phase when Ku expression is turned off. Arrest results from cell cycle checkpoint activation due to persistent signaling via the DNA damage response (DDR). Our results point to the telomeres as the most likely source of the DNA damage signal. Inactivation of the DDR makes the Ku complex dispensable for proliferation in this organism. Our findings suggest that in U. maydis, unprotected telomeres arising from Ku depletion are the source of the signal that activates the DDR leading to cell cycle arrest.