Flexible Positioning of the Telomerase-Associated Nuclease Leads to Preferential Elimination of Nontelomeric DNA

Abstract

In addition to a reverse transcriptase activity, telomerase is associated with a DNA endonuclease that removes nucleotides from a primer 3 terminus prior to telomere repeat addition. Here we examine the DNA specificity of the primer cleavage-elongation reaction carried out by the Euplotes crassus telomerase. We show that the primer cleavage activity copurified with the E. crassus telomerase polymerase, indicating that it either is an intrinsic property of telomerase or is catalyzed by a tightly associated factor. Using chimeric primers containing stretches of telomeric DNA that could be precisely positioned on the RNA template, we found that the cleavage site is more flexible than originally proposed. Primers harboring mismatches in dT tracts that aligned opposite nucleotides 37 to 40 in the RNA template were cleaved to eliminate the mismatched residues along with the adjacent 3 sequence. The cleaved product was then elongated to generate perfect telomeric repeats. Mismatches in dG tracts were not removed, implying that the nuclease does not track coordinately with the polymerase active site. Our data indicate that the telomerase-associated nuclease could provide a rudimentary proofreading function in telomere synthesis by eliminating mismatches between the DNA primer and the 5 region of the telomerase RNA template.The ends of linear eukaryotic chromosomes are nucleoprotein structures comprised of simple G-rich DNA repeats and structural proteins that together form a protective cap known as the telomere. The integrity of the telomere complex is essential for genome stability. Broken chromosomes lacking telomeres are detected at DNA damage checkpoints and, if not repaired, lead to cell cycle arrest (13, 38), chromosome fusion, or degradation (12). In most eukaryotes, telomerase is responsible for the synthesis and maintenance of telomeric DNA (15). Telomerase adds telomeric DNA to pre-existing telomere tracts and forms telomeres de novo on broken chromosome ends (reviewed in reference 29).Telomerase is a reverse transcriptase ribonucleoprotein. The telomerase RNA subunits from a variety of different organisms have been characterized (11,16,39,40), and proteins that fit the criteria for telomerase subunits have been isolated from the ciliates Tetrahymena thermophila and Euplotes aediculatus (7, 24) and from yeast and mammals (18,24,31,32). Telomerase catalyzes polymerization of telomeric repeats onto chromosome 3Ј termini by copying a sequence within its RNA subunit. In addition to the RNA templating domain, telomerase protein components also establish contacts with DNA substrates. A protein anchor site in telomerase binds clusters of dG residues upstream of a primer 3Ј terminus and mediates processive elongation by maintaining primer contact during successive rounds of polymerization and primer translocation (17,23,28,30).The telomerases from T. thermophila, Saccharomyces cerevisiae, and Euplotes crassus are associated with nuclease activities in vitro (6,28,33). The nuclease cuts single-stranded DNA primers, rem...

Developmentally Regulated Telomerase Activity Is Correlated with Chromosomal Healing during Chromatin Diminution in Ascaris suum

“…The ability of telomerase to initiate telomere synthesis, without proper annealing of the 3Ј nucleotide, may also be important for ciliated protozoans during developmentally programmed new telomere addition (75). In vitro, telomere synthesis onto nontelomeric primers occurs by two activities of the ciliate telomerase enzyme: either by direct extension with a predominant permutation of the telomeric sequence (42,68) or by cleavage-initiated extension (12,21,42). The in vivo studies with A. suum have shown no evidence for de novo telomere formation by such mechanisms.…”

Characterization of the Interaction between the Nuclease and Reverse Transcriptase Activity of the Yeast Telomerase Complex

“…Genes encoding the RNA and RT subunit of the enzyme complex have also been cloned for many known telomerases (2,3,5,8,16,18,24,26,34). In addition, both biochemical and genetic studies point to the existence of additional protein subunits of telomerase, whose functions remain to be elucidated (7,9,15,19,27).A telomerase-associated nuclease has been identified in Tetrahymena thermophila, Euplotes crassus, Saccharomyces cerevisiae, and Schizosaccharomyces pombe (4,6,10,20,21,23,29). In the case of Tetrahymena telomerase, the associated nuclease has been found to remove one or several terminal primer nucleotides prior to polymerization.…”

Expression of Mutated Paramecium Telomerase RNAs In Vivo Leads to Templating Errors That Resemble Those Made by Retroviral Reverse Transcriptase

“…Nucleolytic activities associated with both the Tetrahymena and the equally precise Euplotes enzyme (14,42) have been postulated to be part of a proofreading system, in which misincorporated nucleotides are removed from de novo repeats prior to elongation (25). It is conceivable that telomerase from most Paramecium species lack an efficient mechanism to prevent or correct TTP misincorporation, resulting in a high percentage of G 3 T 3 errors.…”

“…This activity either is an intrinsic property of telomerase or is catalyzed by a factor that remains tightly associated with telomerase through extensive purification. It has been postulated that the in vitro endonucleolytic removal of nontelomeric sequences from primers by telomerase may be part of a proofreading system in which mismatched nucleotides are removed prior to elongation (25). Such a proofreading mechanism may have evolved to ensure high fidelity for telomerase, since deviation from species-specific telomeric repeats can have severe consequences on cell viability and nuclear division (31,38,41,48,54).…”