Abstract

Human telomerase functions in maintaining genome stability by adding telomeric repeats to the termini of linear chromosomes. Past studies have revealed profound insights into telomerase functions. However, low abundance of functional telomerase and difficulty in quantifying its activity leave partially characterized its thermodynamic and kinetic properties. Using a newly developed method to count individual extension products, we demonstrate that human telomerase holoenzymes contain fast- and slow-acting catalytic sites. Surprisingly, both active sites become inactive after two consecutive rounds of catalysis. The fast active sites turn off ~40-fold quicker than the slow ones and exhibit higher affinity to substrates. In dimeric enzymes, the two sites work in tandem with the faster site functioning before the slower one. In monomeric enzymes, the active sites also perform single-run catalysis. Interestingly, the inactive enzymes can be reactivated by intracellular telomerase-activating factors (iTAFs) available in multiple cell types. Together, the single-run catalysis and the iTAF-triggered reactivation serve as a novel control circuit to ensure that the telomerase holoenzymes are dynamically controlled to match their number of active sites with the number of telomeres they extend. Such exquisite kinetic control of telomerase activity is expected to play important roles in cell division and ageing.