The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment

Abstract

Significance Telomerase directs the synthesis of new telomeric repeats at chromosome ends, enabling cells to overcome the end replication problem and to continue to divide. The shelterin protein TPP1 interacts with telomerase, promoting both telomerase recruitment and processivity (the addition of multiple telomeric repeats after a single substrate binding event). Here, we identify separation-of-function mutants of TPP1 that eliminate telomerase processivity but leave the telomerase recruitment function intact. When introduced into human cells in a homozygous manner, these mutations can induce critical telomere shortening and cellular senescence. Our observations therefore demonstrate that TPP1 stimulation of telomerase processivity is a key regulatory step in vivo for telomerase-mediated extension of telomeres and continued cell proliferation. The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214–218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.