Structural and Functional Insights into Telomerase Recruitment

Abstract

Telomeres are nucleoprotein complexes that cap the ends of linear chromosomes and preserve genomic integrity. They help overcome two biological problems posed by linear chromosomes. The first problem, called the end protection problem, is solved by the shelterin complex. Shelterin is the six-protein complex that forms the major protein constituent of telomeres, coating telomeric DNA to protect chromosome ends from being misrecognized by the DNA damage response and repair machineries. The shelterin component TPP1 also helps solve the second problem associated with linear chromosomes called the end replication problem, which arises because replicative polymerases cannot fully synthesize DNA at chromosome ends. TPP1 recruits the ribonucleoprotein (RNP) enzyme telomerase to telomeres, where it compensates for telomere attrition by addition of telomeric repeats, thereby enabling continued cell division in stem and germ line cells. The lack of telomerase in most somatic cells limits their unregulated division. However telomerase is upregulated in ~90% of cancers, qualifying it as an attractive target for anti-cancer drug design. Mutations in telomere and telomerase associated genes can lead to a variety of telomeropathies, the most prominent of which is an inherited bone marrow failure syndrome called dyskeratosis congenita (DC). As a consequence of these mutations, patients with DC have extremely short telomeres. Our lab was involved in characterizing DC in a patient who was heterozygous for an in frame deletion of lysine 170 (K170) in the shelterin protein TPP1. Although K170 is found near a region of residues in TPP1 responsible for recruiting telomerase, referred to as the TEL patch, this residue was not identified in previous screens. Here, using a combination of biochemistry and cell biology, we demonstrate that this mutation causes a defect in the ability of TPP1 to recruit telomerase or stimulate its repeat addition processivity, and affects its ability to maintain telomere length in vivo. Using X-ray crystallography, I illustrate that TPP1 K170Δ alters the TEL patch, spatially displacing two critical glutamates. Finally, we use CRISPR-Cas9 gene editing to show that introducing this mutation in a heterozygous context is sufficient to cause telomere shortening in human cells, thereby providing important insights into the genetics of DC. While the surface on TPP1 that is responsible for telomerase recruitment has been well characterized, it is not as clear which regions of the protein subunit of telomerase (TERT) are important for this interaction. The TEN domain and the IFD of TERT have been implicated in TPP1 binding, although only one specific interaction between the TEN domain and the TEL patch has been identified. Here, I use an alanine scanning mutagenesis screen in conjunction with an in-cell recruitment assay to identify one region each in the TEN domain and IFD that contain residues necessary for telomerase recruitment to the telomere. These mutations showed wild-type telomerase RNP assembly but hindered TPP1-stimulated telomerase activity in vitro and adversely affected the extension of telomeres by telomerase in cultured cells. In summary, my studies have helped pinpoint the TERT side the TPP1-telomerase interface that is critical for both cancer cell proliferation and normal stem cell function.