Flexible positioning of the telomerase-associated nuclease leads to preferential elimination of nontelomeric DNA.

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 3′ 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 3′ 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, removing nucleotides from the 3′ terminus prior to primer elongation by telomerase. Although initially observed with telomeric primers (6), the nuclease activity from E. crassus was subsequently shown to eliminate long stretches of 3′ nontelomeric DNA from primers that carry a short internal tract of telomeric sequence (28). Nontelomeric DNA regions as long as 29 nucleotides can be removed by this mechanism (29a). Experiments with the Tetrahymena and Euplotes enzymes suggested that the nuclease active site is fixed relative to the 5′ boundary of the RNA template (1, 6, 28) such that any primer nucleotides extending across this site are subject to elimination. Studies with methylphosphonate-substituted oligonucleotide substrates revealed that cleavage proceeds by an endonucleolytic mechanism (28). The function of the telomerase nuclease activity has been unclear. The combined endonucleolytic and polymerization activities of telomerase could serve a specialized function in ciliated protozoa during site-specific chromosome fragmentation and de novo telomere formation. These events are developmentally programmed and temporally coupled during the formation of the new macronuclear genome following conjugation (reviewed in reference 8). It has also been postulated that cleavage serves a proofreading function (6, 28, 33) and may be mechanistically linked to processivity (5). Here we examine the DNA specificity of the cleavage-initiated polymerization reaction by the Euplotes telomerase. We show that the nuclease activity remains associated with telomerase through extensive purification, indicating that it either is an intrinsic property of the enzyme or is catalyzed by a tightly associated factor. We also demonstrate that the nuclease active site is not rigidly fixed relative to the telomerase RNA. Instead, any primer nucleotides that cannot form Watson-Crick base pairs with the rA residues adjacent to the 5′ boundary of the telomerase RNA templating domain, even single-nucleotide mismatches, are eliminated before synthesis initiates.