Structural and functional consequences of a disease mutation in the telomere protein TPP1.

Telomerase replicates chromosome ends to facilitate continued cell division. Mutations that compromise telomerase function result in stem cell failure diseases, such as dyskeratosis congenita (DC). One such mutation (K170Δ), residing in the telomerase-recruitment factor TPP1, provides an excellent opportunity to structurally, biochemically, and genetically dissect the mechanism of such diseases. We show through site-directed mutagenesis and X-ray crystallography that this TPP1 disease mutation deforms the conformation of two critical amino acids of the TEL [TPP1's glutamate (E) and leucine-rich (L)] patch, the surface of TPP1 that binds telomerase. Using CRISPR-Cas9 technology, we demonstrate that introduction of this mutation in a heterozygous manner is sufficient to shorten telomeres in human cells. Our findings rule out dominant-negative effects of the mutation. Instead, these findings implicate reduced TEL patch dosage in causing telomere shortening. Our studies provide mechanistic insight into telomerase-deficiency diseases and encourage the development of gene therapies to counter such diseases.

The N Terminus of the OB Domain of Telomere Protein TPP1 Is Critical for Telomerase Action.

Telomerase recruitment to telomeres and enzymatic processivity are mediated by TPP1, an essential component of telomere integrity and telomerase function. A surface on the OB domain of TPP1 called the TEL patch is critical for TPP1's telomerase-associated functions. Here, we identify a separate region in the N terminus of the OB domain (termed NOB) of TPP1 that, like the TEL patch, is essential for telomerase repeat addition processivity in vitro as well as telomerase recruitment to telomeres and telomere lengthening in cells. Although well-conserved among most mammalian TPP1 homologs, the NOB region in mice is distinct. Swapping the sequence of human NOB into mouse TPP1 allows it to stimulate human telomerase, qualifying NOB as an important determinant of species specificity for TPP1-telomerase interaction. Our studies show that TPP1 NOB is critical for telomerase function and demonstrate that the telomerase interaction surface on TPP1 is more elaborate than previously appreciated.

Contributions of the TEL-patch amino acid cluster on TPP1 to telomeric DNA synthesis by human telomerase.

Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch--a cluster of amino acids on the surface of the shelterin component TPP1--is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment--binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.