Telomerase regulatory subunit Est3 in two Candida species physically interacts with the TEN domain of TERT and telomeric DNA

Abstract

The yeast telomerase regulatory protein Est3 is required for telomere maintenance in vivo, and shares intriguing structural and functional similarities with the mammalian telomeric protein TPP1. Here we report our physical and functional characterizations of Est3 homologues from Candida parapsilosis and Lodderomyces elongisporus, which bear unique N-and C-terminal tails in addition to a conserved central OB fold. We show that these Est3 homologues form stable complexes with the TEN domain of telomerase reverse transcriptase. Efficient complex formation requires both the N-and C-terminal tails, as well as conserved OB fold residues of Est3. Other Est3 homologues devoid of the tails failed to interact strongly with the cognate TEN domains. Remarkably, the C. parapsilosis Est3 alone exhibits no appreciable DNA-binding activity, but can be crosslinked to telomeric DNA in the presence of the TEN domain. A conserved basic residue on the putative DNAbinding surface of CpEst3 is required for efficient crosslinking. Mutating the equivalent residue in Candida albicans Est3 caused telomere attrition. We propose that interaction with the TEN domain unmasks a functionally important nucleic acid-binding activity in Est3. Our findings provide insights on the mechanisms and evolution of a widely conserved and functionally critical telomeric/ telomerase component.

Specificity Requirements for Human Telomere Protein Interaction with Telomerase Holoenzyme

“…The conservation of an interaction interface between Est3/TPP1 and holoenzyme-assembled TERT is striking given the divergence of human and yeast telomerases overall (5). It is possible that the TPP1 OBD interacts with DNA in addition to TERT, as suggested for Est3 proteins from certain yeasts (19), perhaps dependent on an elongation-specific holoenzyme conformation.…”

“…Studies of the yeast TPP1 ortholog, Est3, are illuminating as precedent for this potential TPP1 function. Est3 interacts directly with yeast TERT (Est2), which can be recapitulated using recombinant Est3 and Est2 TEN domain (18,19). Est3 side chain substitutions guided by modeling with the TPP1 OB-fold structure (20) have led to the definition of Est3 sequence requirements for Est2 interaction and telomerase catalytic activation in vitro and in vivo (18,21,22).…”

Roles of Telomerase Reverse Transcriptase N-terminal Domain in Assembly and Activity of Tetrahymena Telomerase Holoenzyme

“…We suggest as a working model that one or more subunits of Tetrahymena TASC, the human protein TPP1, and the yeast protein Est3 are architecturally and functionally analogous in their respective telomerase holoenzymes: holoenzyme protein interaction(s) with the TEN domain contribute allosteric stabilization of the elongation-competent architecture of the catalytic core RNP and could also extend the direct surface of DNA contact (32,33). Better understanding of the elongating telomerase holoenzyme architecture remains a critical goal for future studies.…”

Biogenesis of telomerase ribonucleoproteins

“…3) and S. castelli (Lee et al 2010;Talley et al 2011). A study of recombinant Est3 from yeast lacking a known Est1 demonstrated crosslinking to single-stranded telomeric DNA dependent on Est3 interaction with the Est2 TEN domain (Yen et al 2011). Incorporation of Est3 into the S. cerevisiae or C. albicans telomerase holoenzyme has a variably reported dependence on Est1 (Osterhage et al 2006;Hsu et al 2007;Lee et al 2010;Tuzon et al 2011).…”