Jayakrishnan Nandakumar

Finding the end: recruitment of telomerase to telomeres

Telomeres, the ends of linear eukaryotic chromosomes, are characterized by the presence of multiple repeats of a short DNA sequence. This telomeric DNA is protected from illicit repair by telomere-associated proteins, which in mammals form the shelterin complex. Replicative polymerases are unable to synthesize DNA at the extreme ends of chromosomes, but in unicellular eukaryotes such as yeast and in mammalian germ cells and stem cells, telomere length is maintained by a ribonucleoprotein enzyme known as telomerase. Recent work has provided insights into the mechanisms of telomerase recruitment to telomeres, highlighting the contribution of telomere-associated proteins, including TPP1 in humans, Ccq1 in Schizosaccharomyces pombe and Cdc13 and Ku70–Ku80 in Saccharomyces cerevisiae.

The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity

Human chromosome ends are capped by shelterin, a protein complex that protects the natural ends from being recognized as sites of DNA damage and also regulates the telomere-replicating enzyme, telomerase. Shelterin includes the heterodimeric POT1–TPP1 protein, which binds the telomeric single-stranded DNA tail. TPP1 has been implicated both in recruiting telomerase to telomeres and in stimulating telomerase processivity (the addition of multiple DNA repeats after a single primer-binding event). Determining the mechanisms of these activities has been difficult, especially because genetic perturbations also tend to affect the essential chromosome end-protection function of TPP1 (refs 15, 16, 17). Here we identify separation-of-function mutants of human TPP1 that retain full telomere-capping function in vitro and in vivo, yet are defective in binding human telomerase. The seven separation-of-function mutations map to a patch of amino acids on the surface of TPP1, the TEL patch, that both recruits telomerase to telomeres and promotes high-processivity DNA synthesis, indicating that these two activities are manifestations of the same molecular interaction. Given that the interaction between telomerase and TPP1 is required for telomerase function in vivo, the TEL patch of TPP1 provides a new target for anticancer drug development.

Hoyeraal-Hreidarsson syndrome caused by a germline mutation in the TEL patch of the telomere protein TPP1

Germline mutations in telomere biology genes cause dyskeratosis congenita (DC), an inherited bone marrow failure and cancer predisposition syndrome. DC is a clinically heterogeneous disorder diagnosed by the triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia; Hoyeraal-Hreidarsson syndrome (HH), a clinically severe variant of DC, also includes cerebellar hypoplasia, immunodeficiency, and intrauterine growth retardation. Approximately 70% of DC cases are associated with a germline mutation in one of nine genes, the products of which are all involved in telomere biology. Using exome sequencing, we identified mutations in Adrenocortical Dysplasia Homolog (ACD) (encoding TPP1), a component of the telomeric shelterin complex, in one family affected by HH. The proband inherited a deletion from his father and a missense mutation from his mother, resulting in extremely short telomeres and a severe clinical phenotype. Characterization of the mutations revealed that the single-amino-acid deletion affecting the TEL patch surface of the TPP1 protein significantly compromises both telomerase recruitment and processivity, while the missense mutation in the TIN2-binding region of TPP1 is not as clearly deleterious to TPP1 function. Our results emphasize the critical roles of the TEL patch in proper stem cell function and demonstrate that TPP1 is the second shelterin component (in addition to TIN2) to be implicated in DC.

How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA

The POT1-TPP1 heterodimer, the major telomere-specific single-stranded DNA-binding protein in mammalian cells, protects chromosome ends and contributes to the regulation of telomerase. The recent discovery of telomeric RNA raises the question of how POT1 faithfully binds telomeric ssDNA and avoids illicit RNA binding that could result in its depletion from telomeres. Here we show through binding studies that a single deoxythymidine in a telomeric repeat dictates the DNA versus RNA discrimination by human POT1 and mouse POT1A. We solve the crystal structure of hPOT1 bound to DNA with a ribouridine in lieu of the critical deoxythymidine and show that this substitution results in burying the 2′-hydroxyl group in a hydrophobic region (Phe62) of POT1 in addition to eliminating favorable hydrogen-bonding interactions at the POT1–nucleic acid interface. At amino acid 62, Phe discriminates against RNA binding and Tyr allows RNA binding. We further show that TPP1 greatly augments POT1’s discrimination against RNA.

Inhibition of Telomerase Recruitment and Cancer Cell Death

Background: A specific surface of the TPP1 protein, called the TEL patch, mediates telomerase recruitment in human cells. Results: TEL patch deficiency caused cancer cell death, which was exacerbated by inhibition of telomerase enzymatic activity. Conclusion: TEL patch mutations are detrimental to cancer cell viability. Significance: This work encourages the development of new combinational cancer therapies that involve simultaneous inhibition of telomerase recruitment and telomerase activity. Continued proliferation of human cells requires maintenance of telomere length, usually accomplished by telomerase. Telomerase is recruited to chromosome ends by interaction with a patch of amino acids (the TEL patch, for TPP1 glutamate (E) and leucine (L)-rich patch) on the surface of telomere protein TPP1. In previous studies, interruption of this interaction by mutation prevented telomere extension in HeLa cells, but the cell culture continued to grow. We now show that the telomerase inhibitor BIBR1532 acts together with TEL patch mutations to inhibit the growth of HeLa cell lines and that apoptosis is a prominent mechanism of death of these cells. Survivor cells take over the population beginning around 40 days in culture. These cells no longer express the TEL patch mutant TPP1, apparently because of silencing of the expression cassette, a survival mechanism that would not be available to cancer cells. These results provide hope that inhibiting the binding of telomerase to the TEL patch of TPP1, perhaps together with a modest inhibition of the telomerase enzyme, could comprise an effective anticancer therapy for the ∼90% of human tumors that are telomerase-positive.

Dual Mechanisms whereby a Broken RNA End Assists the Catalysis of Its Repair by T4 RNA Ligase 2

T4 RNA ligase 2 (Rnl2) efficiently seals 3′-OH/5′-PO4RNA nicks via three nucleotidyl transfer steps. Here we show that the terminal 3′-OH at the nick accelerates the second step of the ligase pathway (adenylylation of the 5′-PO4 strand) by a factor of 1000, even though the 3′-OH is not chemically transformed during the reaction. Also, the terminal 2′-OH at the nick accelerates the third step (attack of the 3′-OH on the 5′-adenylated strand to form a phosphodiester) by a factor of 25–35, even though the 2′-OH is not chemically reactive. His-37 of Rnl2 is uniquely required for step 3, providing a ∼102 rate acceleration. Biochemical epistasis experiments show that His-37 and the RNA 2′-OH act independently. We conclude that the broken RNA end promotes catalysis of its own repair by Rnl2 via two mechanisms, one of which (enhancement of step 3 by the 2′-OH) is specific to RNA ligation. Substrate-assisted catalysis provides a potential biochemical checkpoint during nucleic acid repair.

Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme

Programmed RNA breakage is an emerging theme underlying cellular responses to stress, virus infection and defense against foreign species. In many cases, site-specific cleavage of the target RNA generates 2′,3′ cyclic phosphate and 5′-OH ends. For the damage to be repaired, both broken ends must be healed before they can be sealed by a ligase. Healing entails hydrolysis of the 2′,3′ cyclic phosphate to form a 3′-OH and phosphorylation of the 5′-OH to form a 5′-PO4. Here, we demonstrate that a polynucleotide kinase-phosphatase enzyme from Clostridium thermocellum (CthPnkp) can catalyze both of the end-healing steps of tRNA splicing in vitro. The route of tRNA repair by CthPnkp can be reprogrammed by a mutation in the 3′ end-healing domain (H189D) that yields a 2′-PO4 product instead of a 2′-OH. Whereas tRNA ends healed by wild-type CthPnkp are readily sealed by T4 RNA ligase 1, the H189D enzyme generates ends that are spliced by yeast tRNA ligase. Our findings suggest that RNA repair enzymes can evolve their specificities to suit a particular pathway.

DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe

Eukaryotic chromosome ends are protected from illicit DNA joining by protein–DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA.

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

Significance Telomerase is an enzyme that replicates chromosome ends to facilitate continued stem cell division. Mutations in telomerase or in telomerase-related genes result in stem cell-dysfunction diseases, such as dyskeratosis congenita (DC). Despite its devastating nature, DC currently has no cure. Here we report the crystal structure of a mutant protein implicated in DC to reveal how the mutation disrupts a region of the protein essential for telomerase function. Furthermore, we demonstrated that this mutation, when introduced into a human cell line, is sufficient to cause the cellular underpinnings of DC. Our results therefore make the strong prediction that correcting the mutation in the stem cells of the patient will reverse the cellular symptoms of disease. 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.

Pot1 OB-fold mutations unleash telomere instability to initiate tumorigenesis.

Chromosomal aberrations are a hallmark of human cancers, with complex cytogenetic rearrangements leading to genetic changes permissive for cancer initiation and progression. Protection of Telomere 1 (POT1) is an essential component of the shelterin complex and functions to maintain chromosome stability by repressing the activation of aberrant DNA damage and repair responses at telomeres. Sporadic and familial mutations in the oligosaccharide-oligonucleotide (OB) folds of POT1 have been identified in many human cancers, but the mechanism underlying how hPOT1 mutations initiate tumorigenesis has remained unclear. Here we show that the human POT1’s OB-folds are essential for the protection of newly replicated telomeres. Oncogenic mutations in hPOT1 OB-fold fail to bind to single-stranded telomeric DNA, eliciting a DNA damage response at telomeres that promote inappropriate chromosome fusions via the mutagenic alternative non-homologous end joining (A-NHEJ) pathway. hPOT1 mutations also result in telomere elongation and the formation of transplantable hematopoietic malignancies. Strikingly, conditional deletion of both mPot1a and p53 in mouse mammary epithelium resulted in development of highly invasive breast carcinomas and the formation of whole chromosomes containing massive arrays of telomeric fusions indicative of multiple breakage-fusion-bridge cycles. Our results reveal that hPOT1 OB-folds are required to protect and prevent newly replicated telomeres from engaging in A-NHEJ mediated fusions that would otherwise promote genome instability to fuel tumorigenesis.