dGTP-dependent processivity and possible template switching of euplotes telomerase.

We have measured the processivity of telomeric DNA extension by Euplotes aediculatus telomerase at various concentrations of the nucleotide substrates dGTP and dTTP. The maximum processivity (approximately 3 repeats) was observed at approximately 100 microM of each dNTP. Processivity decreased as the dNTP concentrations were reduced and, surprisingly, as the concentration of dGTP was increased. Also, the characteristic banding pattern generated by telomerase extension of DNA primers shifted in response to changes in dGTP concentration. One pattern with 8 nt periodicity was predominant at dGTP concentrations </=16 microM, while at >/= 250 microM an 8 nt repeat pattern out-of-phase with the first was observed; at intermediate concentrations the two patterns coexisted. We propose that two different segments of the RNA subunit can serve as the template for repeat synthesis; nt 42-49 at low dGTP concentrations and nt 36-43 at high dGTP concentrations. An alternative model for the low dGTP pattern involves an internal pause site but no pause at the end of the template and is, therefore, considered less likely. Because the effects of dGTP on processivity and banding pattern appear to be distinct from nucleotide binding in the polymerase active site, we propose a second dGTP binding site involved in template selection and processivity.     

POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation

Telomerase contributes to chromosome end replication by synthesizing repeats of telomeric DNA, and the telomeric DNA‐binding proteins protection of telomeres (POT1) and TPP1 synergistically increase its repeat addition processivity. To understand the mechanism of increased processivity, we measured the effect of POT1–TPP1 on individual steps in the telomerase reaction cycle. Under conditions where telomerase was actively synthesizing DNA, POT1–TPP1 bound to the primer decreased primer dissociation rate. In addition, POT1–TPP1 increased the translocation efficiency. A template‐mutant telomerase that synthesizes DNA that cannot be bound by POT1–TPP1 exhibited increased processivity only when the primer contained at least one POT1–TPP1‐binding site, so a single POT1–TPP1–DNA interaction is necessary and sufficient for stimulating processivity. The POT1–TPP1 effect is specific, as another single‐stranded DNA‐binding protein, gp32, cannot substitute. POT1–TPP1 increased processivity even when substoichiometric relative to the DNA, providing evidence for a recruitment function. These results support a model in which POT1–TPP1 enhances telomerase processivity in a manner markedly different from the sliding clamps used by DNA polymerases.     

A possible mechanism of processive nucleotide and repeat additions by the telomerase

Telomerase is a specialized cellular ribonucleoprotein complex that can synthesize long stretches of a DNA primer by using an intrinsic RNA template sequence. This requires that the telomerase must be able to carry out both nucleotide and repeat additions. Here, based on available structures and experimental data, a model is presented to describe these two addition activities. In the model, the forward movement of the polymerase active site along the template during the processive nucleotide addition is rectified through the incorporation of a matched base, via the Brownian ratchet mechanism. The unpairing of the DNA:RNA hybrid and then repositioning of product 3′-end after each round of repeat synthesis, which are prerequisites for the processive repeat addition, are caused by a force acting on the primer. The force results from the conformational transition of the stem III pseudoknot, which is mechanically induced by the rotation of TERT fingers together with stem IV loop towards the polymerase active site upon a nucleotide binding. Based on the model, the dynamics of processive nucleotide and repeat additions by recombinant Tetrahymena telomerase is studied analytically, which gives good quantitative explanations to the previous experimental results. Moreover, some predicted results are presented. In particular, it is shown that the repeat addition processivity is mainly determined by the difference between the free-energy change required to disrupt the DNA:RNA hybrid and that required to unfold the stem III pseudoknot. A large difference in free energy corresponds to a low repeat addition processivity while a small difference in free energy corresponds to a high repeat addition processivity.     

Direct involvement of the TEN domain at the active site of human telomerase

Telomerase is a ribonucleoprotein that adds DNA to the ends of chromosomes. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is important for activity and processivity. Here we describe a mutation in the TEN domain of human TERT that results in a greatly increased primer K(d), supporting a role for the TEN domain in DNA affinity. Measurement of enzyme kinetic parameters has revealed that this mutant enzyme is also defective in dNTP polymerization, particularly while copying position 51 of the RNA template. The catalytic defect is independent of the presence of binding interactions at the 5'-region of the DNA primer, and is not a defect in translocation rate. These data suggest that the TEN domain is involved in conformational changes required to position the 3'-end of the primer in the active site during nucleotide addition, a function which is distinct from the role of the TEN domain in providing DNA binding affinity.     

Multiple Mechanisms for Elongation Processivity within the Reconstituted Tetrahymena Telomerase Holoenzyme

To maintain telomeres, telomerase evolved a unique biochemical activity: the use of a single-stranded RNA template for the synthesis of single-stranded DNA repeats. High repeat addition processivity (RAP) of the Tetrahymena telomerase holoenzyme requires association of the catalytic core with the telomere adaptor subcomplex (TASC) and an RPA1-related subunit (p82 or Teb1). Here, we used DNA binding and holoenzyme reconstitution assays to investigate the mechanism by which Teb1 and TASC confer high RAP. We show that TASC association with the recombinant telomerase catalytic core increases enzyme activity. Subsequent association of the Teb1 C-terminal domain with TASC confers the capacity for high RAP even though the Teb1 C-terminal domain does not provide a high-affinity DNA interaction site. Efficient RAP also requires suppression of nascent product folding mediated by the central Teb1 DNA-binding domains (DBDs). These sequence-specific high-affinity DBDs of Teb1 can be functionally substituted by the analogous DBDs of Tetrahymena Rpa1 to suppress nascent product folding but only if the Rpa1 high-affinity DBDs are physically tethered into holoenzyme context though the Teb1 C-terminal domain. Overall, our findings reveal multiple mechanisms and multiple surfaces of protein-DNA and protein-protein interaction that give rise to elongation processivity in the synthesis of a single-stranded nucleic acid product.     

A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity

The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase.