Chem., 294(30):11579-11588, 2019). 2010;11:171181. Because RNA is more vulnerable to hydrolysis than DNA, inserted ribonucleotides are more likely to hydrolyze, which would cause a telomeric single strand break (Baumann and Cech, 2001; de Lange, 2005; Li and Breaker, 1999). The most novel finding was a steric gate which allows TERT to discriminate dNTPs from rNTPs and the authors show that this residue (Y717) serves similar functions in human telomerase. These include premature aging, idiopathic pulmonary fibrosis (IPF), dyskeratosis congenita, and cancer (Blasco, 2005; Kim et al., 1994; Nelson and Bertuch, 2012). Please see changes to the Results section. Garforth SJ, Wu YY, Prasad VR. An official website of the United States government. (C) Eventual consequences of rNTP insertion by telomerase. To address this, we have created a new table further entailing the differences between several different TERTs and added text discussing these crucial differences. We found that, in terms of global protein structure, minimal rearrangement is required to proceed through the catalytic cycle. The transition between these two states represents the nucleotide binding step, measured as a dissociation constant (Kd). Please provide some background on that (perhaps in the Introduction). Cells were grown to 90% confluency in Dulbeccos modified Eagles medium (Gibco) supplemented with 10% High Quality FBS (Hyclone) and 1% penicillin-streptomycin (Corning) at 37C and 5% CO2. Data was fit to Equation 1 (Supplementary file 3, Table 3a and b). Pre-steady-state parameters were also calculated for (B) tcTERT with a Q308A substitution. CST complex is a heterotrimeric protein consisting of conserved telomere protection component 1 (CTC1), suppressor of cdc13 a (STN1), and telomeric pathway with STN1 (TEN1), which specifically localize to the single-stranded 3 overhang and protect the telomeres by mediating DNA replication and telomerase regulation, independent of shelterin complex. So far the structures and kinetics have only been done with one single addition of dGTP in this work, which will be different for other nucleotides of the repeat. Epub 2013 Jun 24. These include tcTERT lacking the Nterminal (TEN) domain and missing a portion of the insertion in fingers domain (IFD) (Supplementary file 1table 1B). A single nucleotide incorporation step limits human telomerase repeat addition activity. The distance from the C2 carbon of the dGpCpp to residue 256 shifted from 3.3 in WT TERT to 5.7 in TERT Y256A, showing that the TERT Y256A has much more room to accommodate the 2-OH (Figure 4J). -, Toupance S., Villemonais D., Germain D., Gegout-Petit A., Albuisson E., Benetos A. While the downstream consequences of telomeric mismatches have not been studied in a biological context to our knowledge, they likely would disrupt G-quadruplex stability and inhibit shelterin protein binding, as both of these phenomena are dependent on DNA sequence (Figure 6B) (Burge, et al., 2006; de Lange, 2005)., [T]elomerase inserts ~40 rNTPs, which represents selectivity comparable to DNA polymerase and DNA polymerase (Brown and Suo, 2011; Cavanaugh, Beard and Wilson, 2010; McElhinny et al., 2010). Schneider CA, Rasband WS, Eliceiri KW. Olovnikov AM. This lack of rearrangement contrasts many other DNA polymerases and even HIV RT, which have been shown to undergo global shifts from an open to a closed state upon nucleotide binding (Doubli et al., 1999; Sawaya et al., 1994; Schmidt et al., 2018). Therefore, telomerase cannot increase telomere length much past the initial primer terminus, potentially limiting the binding and keeping it engaged to the telomere end (with only three nucleotides base pairing). This article is distributed under the terms of the, GUID:E57E59C2-DB19-45BC-A3BA-BD5BBBCAB34B, GUID:89688C43-B90C-4F37-AF31-64DFBF88901E, GUID:C3E800F3-D353-4DF0-99F2-38513B04433F, GUID:F2FAEBDB-F5DC-4FF1-A7B4-265623B73823, National Institute of General Medical Sciences, National Institute of Environmental Health Sciences. RCSB Protein Data Bank. When we performed the same telomeric extension assay with the steric gate variant (Y717A), we observed that the primer extension was increased 3-fold compared to WT hTERT in the presence of rNTPs (Figure 5C,D). Live cell imaging reveals the dynamics of telomerase recruitment to telomeres. In the plant Arabidopsis thaliana, it is TTTAGGG. overhang length). We observed that for the insertion of dATP opposite a templating rC, the catalytic efficiency starkly decreased compared to dGTP insertion, both at the nucleotide binding and chemistry step. Nandakumar J, Podell ER, Cech TR. This is because the pre-nucleotide bound category is visited 6 times (Figure 1A and Figure 1figure supplement 3, steps A1-6) prior to the telomerase translocation step. Article This G-overhang is . Resolution of our crystal structures was determined using correlation coefficients (CC1/2), with the highest resolution shell containing a CC1/2 value of greater than 0.3 (Supplementary file 2, Table 2a). Resultant tcTERT was concentrated down to 18 mg mL1 prior to crystallography, and stored at 4C (Gillis et al., 2008). Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase. 2020. They distinguish natural chromosome ends from double-stranded breaks in DNA and protect chromosome ends from degradation or end-to-end fusion with other chromosomes. This sequence is usually repeated about 3,000 times and can reach up to 15,000 base pairs in length. Of note, out of all six templating positions, the substrate with six templating bases exhibited altered kinetics compared to the other 5 positions. However, a key question is to know how it stays on and translocates after a full hexameric repeat is added. What was the reason for this? Several pathways that regulate telomere length have . Although the human telomerase holoenzyme is composed of multiple accessory subunits, catalysis is localized to the telomerase reverse transcriptase (TERT) subunit (Nguyen et al., 2018). The euchromatin-associated and heterochromatin-associated histone marks are indicated. Further clarification about both phenomena have been added to the manuscript text and also below for reference. Journal of the American Chemical Society. and JavaScript. All 18 telomerase states that are required to add one telomeric repeat are shown in Figure 1 figure supplement 3 for reference. See Supplementary file 2, Table 2a for more details of these parameters for each dataset. We hypothesize that the inhibition by rGTP is caused by factors in the telomeric sequence. Cells were harvested via centrifugation at 4000 x g until lysis. (B) The tcTERT prenucleotide binary complex. From early next year, we will no longer make accept/reject decisions after peer review. and transmitted securely. The inhibitory effect seemed to depend on the number of rNTPs inserted per repeat, with rGTP presence showing the greatest inhibition. With the Q308A variant, the kpol decreased~60 fold to 0.30 s1 and the Kd for dGTP increased~2 fold to 45 M (Figure 3figure supplement 2B,D). The most important new finding was that there is a steric gate that allows TERT to discriminate dNTPs from rNTPs. This should be mentioned in the Discussion. The reviewers and the reviewing editor agreed that this work is significant because it is not known how telomerase selects the correct nucleotide for insertion into the telomeric repeat from a pool of nucleotide with various sugars and base pairing properties. A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. Please include the details of the telomerase used. 2020. 2020. (C) Closeup view of dGpCpp with a polder OMIT map contoured at =3.0 (green mesh). The structures presented in this manuscript are A6, B6, and C6. (F) A comparison of TERT efficiencies adjusted for cellular nucleotide concentrations. The heterochromatin-associated marks include H4K20me3, H3K9me3, and H3K27me3. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. These gels were run at 700 V, 60 A, and 30 W at 30C in order to separate the reaction product from its substrate. In the case of 8oxoguanine or 8-oxodGTP, pre-existing 8-oxoG in the telomere strand increased telomerase activity by disrupting G-quadruplex structures that prevented telomerase binding, whereas 8-oxodGTP that was inserted by telomerase inhibited the activity of telomerase. PubMed 15/18 structural states of A2 to C6, Figure 1figure supplement 3) as compared to a six-nucleotide overhang that is representative of only 3/18 structural states. Table 3b. Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. For states without a nucleotide triphosphate bound, the position of the active site is shown with a black circle. So far, the only nucleic acid substrate that has successfully yielded structural information was the one featured in this paper. 2, 251262 (2002). (D) Closeup views of the rC binding residues, (E) terminal dG binding residues, (F) nucleoside binding residues, and (G) the tcTERT catalytic residues and triphosphate coordinating residues. The base marked by an asterisk is complementary to the rNTP in the solution. 2018 Nov;57(11):564-572. doi: 10.1002/gcc.22669. Muller named these ends telomeres (from the Greek words telo, meaning. Jiang J, Wang Y, Suac L, Chan H, Basu R, Zhou ZH, Feigon J. https://doi.org/10.1038/s41556-022-00991-z, An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory. Fay MM, Lyons SM, Ivanov P. RNA G-Quadruplexes in biology: principles and molecular mechanisms. Importantly, as the telomerase approaches the end of its template, the DNA:RNA duplex at the 5 end begins to melt, enabling telomerase to either (1) translocate and anneal the RNA component to the newly extended telomeric repeat, thus allowing for additional repeat addition; or (2) dissociate from the telomeric DNA., Our experiments were carried out specifically with a single dGTP insertion using a 4 nucleotide overhang RNA template. Mitchell M, Gillis A, Futahashi M, Fujiwara H, Skordalakes E. Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. In the meantime, to ensure continued support, we are displaying the site without styles ), steady-state kinetics were performed for all six templating positions. It was recently shown telomerase becomes inactive after two repeats, but can be reactivated by the recently discovered intracellular telomerase-activating factors (iTAFs) (Sayed et al., 2019).. Transcribed image text: Eukaryotic Telomeres are usually rich in which nucleotide? In each case, the conditions used for each reaction were: 25 mM TRIS pH 7.5, 0.05 mg mL1 Bovine Serum Albumin, 1 mM dithiothreitol, 10% glycerol, 200 mM KCl, 1 M tcTERT, 100 nM annealed DNA:RNA hybrid substrate, and varying concentrations of the nucleotide triphosphate of interest. ; Illustration showing the position of telomeres at the end of our chromosomes. doi: 10.1038/s41576-019-0099-1. Epub 2023 May 29. Characterization of the telomerase catalytic mechanism was performed using pre-steady-state kinetics of single nucleotide insertion by tcTERT (Figure 3, Figure 3figure supplement 1, Figure 3figure supplement 2, and Supplementary file 3). Structure of telomerase. In a natural telomerase RNP, the RNA template threads through the TERT ring. Structural features of mouse telomerase RNA are responsible for the lower activity of mouse telomerase versus human telomerase. Reactions contained 1x human telomerase buffer, 5 nM of 32P-end-labeled primer and 50 M dNTP or rNTP mix as indicated in the figure legends. Telomeres are sections of DNA found at the ends of each of our chromosomes. A superposition of the tcTERT product structure active site (state C6, yellow) with the active site from the product structure from ttTERT (State C3, green, from Jiang et al., 2018, PDB code: 6D6V). These domains are essential for the activity of other telomerase homologs, and have been hypothesized to be particularly important for telomerase ratcheting during translocation (Steczkiewicz et al., 2011). -, Blackburn E.H., Epel E.S., Lin J. Nelson ND, Bertuch AA. Therefore, we kept our tcTERT kinetics within a single turnover (i.e. Immunol. Nat Cell Biol 24, 14491450 (2022). Bethesda, MD 20894, Web Policies TERRA is involved in regulating telomere capping and the maintenance of telomeres. The active site residues discussed are highly conserved throughout all four species. This site needs JavaScript to work properly. For each of the structures, Ramachandran analysis revealed a minimum of 100% of non-glycine residues occupied allowed regions and at least 93% occupied favored regions. Samples were flash frozen and stored a 80C. Finally, for the product complex, we formed a DNA strand one nucleotide longer by incubating 2.5 mM dGTP with tcTERT and its nucleic acid substrate, allowing the reaction to occur at 22C for 30 min prior to setting up crystallization drops. Prior to crystallization, we complexed tcTERT with its nucleic acid substrate by mixing them at a 1:1.2 ratio of protein to DNA. rGTP displayed the greatest inhibition. For the mismatched insertion, the kpol decreased 129-fold to 0.0081 s1 and the Kd increased 76-fold to 1.3 mM (Figure 3E). This structural agreement between different states and homologs implies that active site contacts are similar throughout all 6 overhang lengths. Although it is unknown why the TERT catalytic core does not open and close, it may be because other complexities necessary for telomerase function limit the opening and closing from occurring, including the translocation step during repeat addition or the extensive interaction with its RNA component.
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