Human Ku70/80 associates physically with telomerase through interaction with hTERT

Telomere length maintenance, an activity essential for chromosome stability and genome integrity, is regulated by telomerase- and telomere-associated factors. The DNA repair protein Ku (a heterodimer of Ku70 and Ku80 subunits) associates with mammalian telomeres and contributes to telomere maintenance. Here, we analyzed the physical association of Ku with human telomerase both in vivo and in vitro. Antibodies specific to human Ku proteins precipitated human telomerase in extracts from tumor cells, as well as from telomerase-immortalized normal cells, regardless of the presence of DNA-dependent protein kinase catalytic subunit. The same Ku antibodies also precipitated in vitro reconstituted telomerase, suggesting that this association does not require telomeric DNA. Moreover, Ku associated with the in vitro translated catalytic subunit of telomerase (hTERT) in the absence of telomerase RNA (hTR) or telomeric DNA. The results presented here are the first to report that Ku associates with hTERT, and this interaction may function to regulate the access of telomerase to telomeric DNA ends.     

A human telomerase-associated nuclease

Ciliate and yeast telomerase possess a nucleolytic activity capable of removing DNA from the 3' end of a single-stranded oligonucleotide substrate. The nuclease activity is thought to assist in enzyme proofreading and/or processivity. Herein, we report a previously uncharacterized human telomerase-associated nuclease activity that shares several properties with ciliate and yeast telomerases. Partially purified human telomerase, either from cell extracts or recombinantly produced, demonstrated an ability to remove 3' nontelomeric nucleotides from a substrate containing 5' telomeric DNA, followed by extension of the newly exposed telomeric sequence. This cleavage/extension activity was apparent at more than one position within the telomeric DNA and was influenced by sequences 5' to the telomeric/nontelomeric boundary and by substitution with a methylphosphonate moiety at the telomeric/nontelomeric DNA boundary. Our data suggest that human telomerase is associated with an evolutionarily conserved nucleolytic activity and support a model in which telomerase-substrate interactions can occur distal from the 3' primer end.     

Mammalian telomeres and telomerase

New features of mammalian telomeres and telomerase have been identified. Telomeres form t-loops, which engage the 3' single-stranded DNA overhang in an interaction with double-stranded telomeric repeats. Mammalian telomerases contain an RNA H/ACA motif and associated protein(s) shared with H/ACA family of small nucleolar ribonucleoproteins. Essential roles for telomerase in the sustained viability of cultured tumor cells and in the normal proliferative capacity of human somatic cells have been demonstrated.     

Tracing the path of DNA substrates in active Tetrahymena telomerase holoenzyme complexes: mapping of DNA contact sites in the RNA subunit

Telomerase, the enzyme that extends single-stranded telomeric DNA, consists of an RNA subunit (TER) including a short template sequence, a catalytic protein (TERT) and accessory proteins. We used site-specific UV cross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA-DNA hybrids made with overlapping complementary oligodeoxynucleotides. These data allowed tracing of the DNA path through the telomerase complexes from the template to the TERT binding element (TBE) region of TER. TBE is known to bind TERT and to be involved in the template 5'-boundary definition. Based on these findings, we propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5'-end of the RNA template. The upstream DNA-TBE interaction may also function as an anchor for the subsequent realignment of the 3'-end of the DNA with the 3'-end of the template to enable initiation of synthesis of a new telomeric repeat.