Studies on the minimal lengths required for DNA primers to be extended by the Tetrahymena telomerase: implications for primer positioning by the enzyme

Baran, Nava; Haviv, Yonit; Paul, Beena; Manor, Haim

doi:10.1093/nar/gkf676

Cited by 23 publications

(25 citation statements)

References 40 publications

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“…2 B-E). This apparent uncoupling of the register of DNA-TERT interaction from the register of DNA-template interaction is consistent with results from our previous DNA footprinting and kinetic assays (10,18,19).…”

Section: Discussionsupporting

confidence: 91%

“…For cleavage of crosslinked complexes, tTERT was immunopurified on FLAG antibody M2 resin (Sigma, St. Louis, MO), as described in ref. 18. Briefly, a reaction mixture of 120-150 l containing crosslinked sample was swirled with 20 l of 1:1 bead slurry for 16 h at 4°C.…”

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confidence: 99%

“…For complete repeat synthesis reactions, this radiolabeled dGTP was supplemented with 2 M extra unlabeled dGTP and 0.2 mM dTTP. Reactions were performed at room temperature, stopped, and assayed by urea polyacrylamide gel electrophoresis (18). Dried gels were imaged and product intensities were quantified by using the Typhoon (GE Healthcare) and ImageQuant software.…”

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confidence: 99%

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High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase

Romi¹,

Baran²,

Gantman³

et al. 2007

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

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Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the K m for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the templateadjacent single-stranded DNA binding site within a cycle of repeat synthesis.specific cleavage of proteins ͉ telomerase-primer interaction ͉ UV crosslinking T elomerase is a unique reverse transcriptase (RT) that extends the single-stranded 3Ј overhangs of telomeres by copying a template within the integral RNA component of the enzyme (1). Some telomerase enzymes can also use this internal template to direct the synthesis of telomeres at nontelomeric sites of chromosome fragmentation (2). In addition to the telomerase RNA subunit (TER), the enzyme contains a catalytic protein subunit, designated telomerase RT (TERT), and accessory proteins (3, 4).Telomerase was first discovered in extracts of the ciliate Tetrahymena thermophila (5), and telomerase from this organism remains an excellent model system for studies of enzyme structure and function. Its RNA subunit (tTER) of 159 nt contains the repeat-complementary sequence 3Ј-AACCCCAAC-5Ј and other motifs required for ribonucleoprotein (RNP) assembly and activity (1, 3). T. thermophila TERT (tTERT) consists of 1,117 amino acids, including a region between residues 518 and 881 that is conserved among RTs and designated as the RT domain (6). The N-terminal half of TERT contains motifs conserved among TERTs but not viral RTs. It constitutes two independently folded domains: the TERT essential N-terminal domain (TEN) and the TERT high-affinity TER binding domain (TRBD). In tTERT, residues 1-195 can be considered to constitute the TEN domain, whereas residues 196-528 comprise the TRBD (7-9).Telomerase specificity of interaction with single-stranded DNA has been studied by monitoring the elongation of primers of varying lengths, sequences and concentrations. Differences in the primer concentration-dependence and repeat addition processivity of product synthesis indirectly suggest that extensive contacts to the enzyme are made by primer regions 5Ј of the template hybrid (2). More direct physical assays have...

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Section: Discussionsupporting

confidence: 91%

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confidence: 99%

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confidence: 99%

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High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase

Romi¹,

Baran²,

Gantman³

et al. 2007

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

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“…the IFD motif) can help to stabilize short RNA-DNA hybrids (33). Indeed, even very short primers (lacking the presumed anchor site target) can mediate multiple repeat addition by Tetrahymena telomerase, albeit rather inefficiently (37). Thus, anchor site interactions may greatly stimulate, but not be essential for, multiple repeat addition.…”

Section: Figmentioning

confidence: 99%

A Physical and Functional Constituent of Telomerase Anchor Site

Lue

2005

Journal of Biological Chemistry

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Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. It consists minimally of a catalytic protein component (TERT) and an RNA subunit that provides the template. Compared with prototypical reverse transcriptases, telomerase is unique in possessing a DNA binding domain (anchor site) that is distinct from the catalytic site. Yeast TERT mutants bearing deletion or point mutations in an N-terminal domain (known as N-GQ) were found to be selectively impaired in extending primers that form short hybrids with telomerase RNA. The mutants also suffered a significant loss of repeat addition processivity but displayed an enhancement in nucleotide addition processivity. Furthermore, the mutants manifested altered primer utilization properties for oligonucleotides containing non-telomeric residues in the 5-region. Crosslinking studies indicate that the N-GQ domain physically contacts the 5-region of the DNA substrate in the context of a telomerase-telomere complex. Together, these results implicate the N-GQ domain of TERT as a physical and functional constituent of the telomerase anchor site. Coupled with previous genetic analysis, our data confirm that anchor site interaction is indeed important for telomerase function in vivo.Telomerase is a ribonucleoprotein that is responsible for maintaining the terminal repeats of telomeres in most organisms (1). It acts as an unusual reverse transcriptase, using a small segment of an integral RNA component as template for the synthesis of the dG-rich strand of telomeres (2).The enzymatic core of telomerase consists minimally of two components, a reverse transcriptase (RT) 1 -like protein that catalyzes nucleotide addition (named TERT), and an RNA in which the template is embedded (for reviews see Refs. 3-6). Telomerase RNAs are evolutionarily divergent in sequence, but recent studies suggest that they share a number of key structural elements, including a template segment, a pseudoknot, and a long-range base-pairing element (for reviews, see Refs. 7 and 8). The TERT protein, on the other hand, is well conserved in evolution and possesses a central domain with significant sequence similarity to prototypical RTs (Fig. 1A) (9, 10). Flanking the RT domain are a large N-terminal extension and a short C-terminal extension (CTE). By analogy with prototypical RTs, the CTE is likely to constitute the so-called thumb domain of the polymerase (11,12). The N-terminal extension consists of a non-conserved region (named N-region), four conserved motifs (named GQ, CP, QFP, and T), and a flexible linker (located between the GQ and CP motif) (13, 14). The CP, QFP, and T motifs have been shown to mediate interaction with telomerase RNA (15-18). The N-region and GQ motif together apparently comprise a stable domain (named N-GQ) that is required for telomerase function both in vitro and in vivo (see below).An unusual property of telomerase is its ability to mediate realignment of the DNA product relative to the RNA templat...

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“…Processive elongation of single-stranded DNA (ssDNA) termini requires that the DNA substrate remain bound to the enzyme while being extended with telomeric repeats. This interaction is thought to be mediated by contacts with one or more primer/product anchor/alignment sites (or PAS) within the TERT protein (2,8,10,21,31). Biochemical and structural analyses suggest that at least one PAS resides within the TEN/ GQ/RID1 domain (25,34,47).…”

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The Telomerase-Specific T Motif Is a Restrictive Determinant of Repetitive Reverse Transcription by Human Telomerase

Drosopoulos

Prasad

2010

Molecular and Cellular Biology

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The central hallmark of telomerases is repetitive copying of a short, defined sequence within its integral RNA subunit. We sought to identify structural determinants of this unique activity in the catalytic protein subunit telomerase reverse transcriptase (TERT) of telomerase. Residues within the highly conserved telomerasespecific T motif of human TERT were mutationally probed, leading to variant telomerases with increased repeat extension rates and wild-type processivity. The extension rate increases were independent of template sequence composition and only moderately correlated to telomerase RNA (TR) binding. Importantly, analysis of substrate primer elongation showed that the extension rate increases primarily resulted from increases in the repeat (type II) translocation rate. Our findings indicate a participatory role for the T motif in repeat translocation, an obligatory event for repetitive telomeric DNA synthesis. Thus, the T motif serves as a restrictive determinant of repetitive reverse transcription.In eukaryotes, chromosomal telomeres serve to maintain genomic integrity, functioning both as buffers against genetic erosion and as suppressors of chromosome fusion and deleterious recombinational events (7). A distinguishing feature of the DNA component of these nucleoprotein structures is the presence of multiple short, tandem repeats. De novo synthesis of telomeric DNA is performed by the cellular polymerase telomerase. This specialized ribonucleoprotein reverse transcriptase (RT) accomplishes this via repetitive copying of a short, defined sequence within its integral RNA subunit (TR) by its catalytic protein subunit, telomerase reverse transcriptase (TERT).Investigations into TERT sequence/function relationships have led to the elucidation of a basic structural framework for the catalytic subunit. The TERT subunit is comprised of a central RT domain with conserved sequence motifs shared with retroviral and other RTs, which is flanked by N-and C-terminal extensions specific to telomerases (Fig. 1). The N-terminal extension (NTE) is further subdivided into two domains, the N-terminal-most TEN (TERT essential N-terminal)/GQ/RID1 (RNA interaction domain-1) domain and the TRBD (TERT RNA binding domain)/RID2 (RNA interaction domain-2) domain, separated by a nonconserved linker. The catalysis of nucleotide addition (polymerization) is performed by the RT domain, while other functional roles have been ascribed to the remaining three telomerase-specific domains. The TEN/GQ/RID1 domain displays binding affinity for single-stranded telomeric DNA as well as low affinity for TR (25,34,39,42). These interactions provide the underlying basis for the implicated important role of the TEN/GQ/RID1 domain in DNA substrate recognition and repeat addition processivity.The TRBD displays a relatively high affinity for TR, and this TERT-TR interaction is thought to promote stable assembly of the ribonucleoprotein complex (4,14,30,38). Among the enzymatic functions linked to the C-terminal extension (CTE) are participation i...

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Studies on the minimal lengths required for DNA primers to be extended by the Tetrahymena telomerase: implications for primer positioning by the enzyme

Cited by 23 publications

References 40 publications

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase

A Physical and Functional Constituent of Telomerase Anchor Site

The Telomerase-Specific T Motif Is a Restrictive Determinant of Repetitive Reverse Transcription by Human Telomerase

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