The Telomerase-Specific T Motif Is a Restrictive Determinant of Repetitive Reverse Transcription by Human Telomerase

Abstract

The central hallmark of telomerases is repetitive copying of a short, defined sequence within its integral RNA subunit. We sought to identify structural determinants of this unique activity in the catalytic protein subunit telomerase reverse transcriptase (TERT) of telomerase. Residues within the highly conserved telomerase-specific T motif of human TERT were mutationally probed, leading to variant telomerases with increased repeat extension rates and wild-type processivity. The extension rate increases were independent of template sequence composition and only moderately correlated to telomerase RNA (TR) binding. Importantly, analysis of substrate primer elongation showed that the extension rate increases primarily resulted from increases in the repeat (type II) translocation rate. Our findings indicate a participatory role for the T motif in repeat translocation, an obligatory event for repetitive telomeric DNA synthesis. Thus, the T motif serves as a restrictive determinant of repetitive reverse transcription.

We thank the AECOM DNA Sequencing Facility.

This work was supported by Public Health Service grants K01 CA87542 (Howard Temin Award) to W.C.D. and R37 AI030861 to V.R.P.