Multiple DNA-binding sites in Tetrahymena telomerase

Finger, Sharon N.; Bryan, Tracy M.

doi:10.1093/nar/gkm866

Cited by 38 publications

(52 citation statements)

References 42 publications

(109 reference statements)

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“…The finding that pseudoknot formation is unnecessary for primer binding is not entirely surprising given that TERT has an RNA-independent DNA-binding activity (40). On the other hand, previous studies showed varying levels of activity defect in pseudoknot substitution and deletion mutants, complicating unambiguous determination of the functional importance of the pseudoknot structure (12,13,(26)(27)(28).…”

Section: Discussionmentioning

confidence: 99%

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Mihalusova¹,

Wu²,

Zhuang

2011

Proc. Natl. Acad. Sci. U.S.A.

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Telomerase ribonucleoprotein (RNP) employs an RNA subunit to template the addition of telomeric repeats onto chromosome ends. Previous studies have suggested that a region of the RNA downstream of the template may be important for telomerase activity and that the region could fold into a pseudoknot. Whether the pseudoknot motif is formed in the active telomerase RNP and what its functional role is have not yet been conclusively established. Using single-molecule FRET, we show that the isolated pseudoknot sequence stably folds into a pseudoknot. However, in the context of the full-length telomerase RNA, interference by other parts of the RNA prevents the formation of the pseudoknot. The protein subunits of the telomerase holoenzyme counteract RNA-induced misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of the pseudoknot but also reveal the critical role played by telomerase proteins in pseudoknot folding.T elomeres shorten with each round of DNA replication. Once telomeres reach a critical length, they become vulnerable to DNA damage response, which triggers cellular senescence and chromosome fusion (1). Telomerase protects telomeres from this replication-induced DNA erosion by addition of short G-rich repeats to the DNA ends (2). The telomerase enzyme is an attractive drug target in both cancer and regenerative medicine, because most highly proliferative cells, including stem and cancer cells, rely on its activity to maintain genomic integrity (3). Furthermore, multiple human diseases are known to be caused by mutations in telomerase subunits (4-6), underscoring the importance of understanding the enzyme's structure and function.The ciliate Tetrahymena thermophila has long been used as a model system to study telomerase activity. Tetrahymena telomerase is comprised of a 159-nucleotide RNA subunit, a 133-kDa telomerase reverse transcriptase (TERT), and multiple protein cofactors (7). The RNA has a conserved secondary structure (Fig. 1A) that contains a template region, which serves as template for the telomeric repeat synthesis, flanked upstream by the template boundary element (TBE) and downstream by a putative pseudoknot, as well as a highly structured region made of stems I and IV (8-10). TERT, the catalytic subunit, binds telomerase RNA in the TBE region, the template recognition element, and the loop of stem IV (10-13) (Fig. 1A) . Of the multiple Tetrahymena telomerase cofactors identified to date, only p65 binds the RNA directly in the stem I-proximal stem IV region (Fig. 1A) and promotes the hierarchical telomerase RNP assembly (14-16).Past studies have suggested that the putative pseudoknot region downstream of the template is a conserved and functionally important segment of telomerase RNA. Phylogenetic analyses of ciliate (9, 17), yeast (18-20), and vertebrate (21) RNAs suggest that this region may fold into a pse...

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Section: Discussionmentioning

confidence: 99%

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Mihalusova¹,

Wu²,

Zhuang

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Notably, there was a higher accumulation of short DNA products in the released fraction (Fig. 3B, Right, T6 trace lines), likely resulting from insufficient interactions between the short DNA product and TERT DNA anchor sites (19,20).…”

Section: Amv Rt)mentioning

confidence: 99%

A self-regulating template in human telomerase

Brown

Podlevsky

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Telomerase is a specialized reverse transcriptase (RT) containing an intrinsic telomerase RNA (TR) component. It synthesizes telomeric DNA repeats, (GGTTAG)n in humans, by reiteratively copying a precisely defined, short template sequence from the integral TR. The specific mechanism of how the telomerase active site uses this short template region accurately and efficiently during processive DNA repeat synthesis has remained elusive. Here we report that the human TR template, in addition to specifying the DNA sequence, is embedded with a single-nucleotide signal to pause DNA synthesis. After the addition of a dT residue to the DNA primer, which is specified by the 49 rA residue in the template, telomerase extends the DNA primer with three additional nucleotides and then pauses DNA synthesis. This sequence-defined pause site coincides precisely with the helix paired region 1 (P1)-defined physical template boundary and precludes the incorporation of nontelomeric nucleotides from residues outside the template region. Furthermore, this sequence-defined pausing mechanism is a key determinant, in addition to the P1-defined template boundary, for generating the characteristic 6-nt ladder banding pattern of telomeric DNA products in vitro. In the absence of the pausing signal, telomerase stalls nucleotide addition at multiple sites along the template, generating DNA products with heterogeneous terminal repeat registers. Our findings demonstrate that this unique self-regulating mechanism of the human TR template is essential for high-fidelity synthesis of DNA repeats.telomeres | ribonucleoprotein | polymerase T he ends of human chromosomes consist of precise repetitions of a 6-nucleotide (nt) sequence synthesized by thespecialized reverse transcriptase (RT), telomerase (1). The telomerase core enzyme is minimally composed of the catalytic telomerase reverse transcriptase (TERT) and the integral telomerase RNA (TR) components (2). Human TR (hTR) is a 451-nt noncoding RNA containing an exceedingly short 11-nt template, which encodes specifically for the telomeric DNA repeat GGTTAG (Fig. 1A, Left). The resulting highly repetitive tract of DNA is bound in a sequence-specific manner by the shelterin complex, which protects natural chromosome termini from endto-end fusions and other DNA damage responses (3, 4). Highfidelity synthesis of telomeric DNA repeats by telomerase is crucial for maintaining telomere function and chromosome stability. Appending the termini of telomeres with even single-nucleotide variations in the telomeric DNA repeat sequence is sufficient for compromising the protective function of the shelterin complex, culminating in deleterious genome instability and cell death (5-8).Whereas TR sequences are highly divergent across taxa, the TR template itself is highly conserved (9, 10). Within vertebrates, the template sequence is conserved with the 5′ boundary defined by a long-range base-paired region known as helix paired region 1 (P1), which constrains and restricts the region that functions as the template f...

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“…By contrast, Type II (repeat addition) processivity describes the ability of telomerase to reposition the 39 end of a newly synthesized repeat within the active site for a second round of reverse transcription (Greider, 1991;Lue, 2004). Repeat addition processivity of both human and Tetrahymena telomerase is affected by sequences at the 59 end of the primer (Finger and Bryan, 2008;Jacobs et al, 2006;Lee and Blackburn, 1993). Multiple experiments have suggested that a region of telomerase outside of the active site mediates an 'anchor-site' interaction with these upstream primer nucleotides that is important for the ability of telomerase to undergo reiterative copying of the RNA template (Autexier and Lue, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…Mutations within the Tetrahymena TEN domain decrease interaction with the DNA primer (Jacobs et al, 2006) and the primer can be photocrosslinked to a fragment containing the Tetrahymena and S. cerevisiae TEN domains (Lue, 2005;Romi et al, 2007). Direct binding assays have demonstrated that the isolated TEN domain from human and Tetrahymena binds telomeric DNA (Finger and Bryan, 2008;Sealey et al, 2010;Wyatt et al, 2007;Wyatt et al, 2009). In S. cerevisiae, a fragment of Est2p containing the TEN domain interacts with full-length TLC1 RNA and increasing amounts of DNA compete with the RNA for protein binding in vitro (Xia et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

Bairley

Guillaume

Vega

et al. 2011

Journal of Cell Science

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SummaryTelomerase is a ribonucleoprotein complex that is required for maintenance of linear chromosome ends (telomeres). In yeast, the Est2 protein reverse transcribes a short template region of the TLC1 RNA using the chromosome terminus to prime replication. Yeast telomeres contain heterogeneous G 1-3 T sequences that arise from incomplete reverse transcription of the TLC1 template and alignment of the DNA primer at multiple sites within the template region. We have previously described mutations in the essential N-terminal TEN domain of Est2p that alter telomere sequences. Here, we demonstrate that one of these mutants, glutamic acid 76 to lysine (est2-LT E76K ), restricts possible alignments between the DNA primer and the TLC1 template. In addition, this mutant exhibits increased processivity in vivo. Within the context of the telomerase enzyme, the Est2p TEN domain is thought to contribute to enzyme processivity by mediating an anchor-site interaction with the DNA primer. We show that binding of the purified TEN domain (residues 1-161) to telomeric DNA is enhanced by the E76K mutation. These results support the idea that the anchor-site interaction contributes to telomerase processivity and suggest a role for the anchor site of yeast telomerase in mediating primer-template alignment within the active site.

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Multiple DNA-binding sites in Tetrahymena telomerase

Cited by 38 publications

References 42 publications

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

A self-regulating template in human telomerase

A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

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