Human telomerase specialization for repeat synthesis by unique handling of primer-template duplex

Abstract

With eukaryotic genome replication, incomplete telomere synthesis results in chromosome shortening and eventual compromise of genome stability. Telomerase counteracts this terminal sequence loss by synthesizing telomeric repeats through repeated cycles of reverse transcription of its internal RNA template. Using human telomerase domain-complementation assays for telomerase reverse transcriptase protein (TERT) and RNA in combination with the first direct footprinting assay for telomerase association with bound DNA, we resolve mechanisms by which TERT domains and RNA motifs direct repeat synthesis. Surprisingly, we find that product-template hybrid is sensed in a length-and sequence-dependent manner to set the template 5 0 boundary. We demonstrate that the TERT N-terminal (TEN) domain determines active-site use of the atypically short primer-template hybrid necessary for telomeric-repeat synthesis. Also against expectation, we show that the remainder of TERT (the TERT ring) supports functional recognition and physical protection of single-stranded DNA adjacent to the template hybrid. These findings establish unprecedented polymerase recognition specificities for DNA-RNA hybrid and single-stranded DNA and suggest a new perspective on the mechanisms of telomerase specialization for telomeric-repeat synthesis.

Dynamics of Human Telomerase Holoenzyme Assembly and Subunit Exchange across the Cell Cycle

“…It is possible that the substantial steady-state pool of hTERT-free hTR RNP (43) is not competent for active RNP assembly. This suggestion is consistent with poor assembly of active RNP by mixing hTERT and hTR cell extracts or separately folded subunits in vitro (58). It will be interesting to apply the in vivo cross-linking approach in combination with competition assays to examine the cell cycle dynamics of telomerase holoenzyme subunit interactions in budding yeast, which is suggested to include disassembly of the catalytic core (52).…”

Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE

“…The arms are entirely dispensable for in vitro activity, since a further-miniaturized allele consisting only of the 170-nt core is also able to reconstitute activity to Mini-T levels (Qiao and Cech 2008), indicating that the core is the only portion required for basal activity. For the human enzyme, in vitro activity can be reconstituted by similarly combining TERT with RNA core nucleotides 33-191, although human telomerase also requires an 87-nt element outside its core, CR4/5, either provided in trans or tethered to the core (Autexier et al 1996;Mitchell and Collins 2000;Wu and Collins 2014).…”

Direct Single-Stranded DNA Binding by Teb1 Mediates the Recruitment of Tetrahymena thermophila Telomerase to Telomeres

“…Extensive studies have demonstrated that within the telomerase catalytic core, both TERT and TER interact with ssDNA. The TERT N-terminal (TEN) domain stimulates active-site use of a short primertemplate hybrid, while the remainder of TERT provides some functional recognition and physical protection of ssDNA (31). Within TER, the template binds to the ssDNA 3= end by hybridization, and other RNA motifs contribute to template placement in the active site (9,31).…”

“…The TERT N-terminal (TEN) domain stimulates active-site use of a short primertemplate hybrid, while the remainder of TERT provides some functional recognition and physical protection of ssDNA (31). Within TER, the template binds to the ssDNA 3= end by hybridization, and other RNA motifs contribute to template placement in the active site (9,31). Additional telomerase holoenzyme proteins augment the activity of the catalytic core in vitro, but the relation of these in vitro activities to their roles in vivo remains unclear.…”