A single nucleotide incorporation step limits human telomerase repeat addition activity

Human telomerase synthesizes telomeric DNA repeats (GGTTAG)_n onto chromosome ends using a short template from its integral telomerase RNA (hTR). However, telomerase is markedly slow for processive DNA synthesis among DNA polymerases. We report here that the unique template-embedded pause signal restricts the first nucleotide incorporation for each repeat synthesized, imparting a significantly greater K_M. This slow nucleotide incorporation step drastically limits repeat addition processivity and rate under physiological conditions, which is alleviated with augmented concentrations of dGTP or dGDP, and not with dGMP nor other nucleotides. The activity stimulation by dGDP is due to nucleoside diphosphates functioning as substrates for telomerase. Converting the first nucleotide of the repeat synthesized from dG to dA through the telomerase template mutation, hTR-51U, correspondingly shifts telomerase repeat addition activity stimulation to dATP-dependent. In accordance, telomerase without the pause signal synthesizes DNA repeats with extremely high efficiency under low dGTP concentrations and lacks dGTP stimulation. Thus, the first nucleotide incorporation step of the telomerase catalytic cycle is a potential target for therapeutic enhancement of telomerase activity.