ИСТИНА

Telomerase is an RNA-proteins complex involved in the elongation of telomeres – special structures at the ends of eukaryotic chromosomes. Telomerase is activated in cancer cells, providing their unlimited division, thus telomerase is a promising target for anti-cancer therapy. Two components provides the activity of telomerase in vitro. Protein catalytic subunit, TERT is a reverse transcriptase that synthetizes the repeat according to template encoded in telomerase RNA (TER). Only low resolution EM structure of human telomerase complex and X-ray, NMR data about separate domains of TERT and TER from different organisms are available. To develop in vitro reconstitution system from purified components of telomerase we took advantage of model organism - the thermos-tolerant yeast, Hansenula polymorpha (hp). Genes for catalytic subunit (hpTERT), telomerase RNA (hpTER) and additional components hpEST1 and hpEST3 were identified. Knockout of any of these genes resulted in telomere shortening. The analysis of HpTER secondary structure revealed all functional elements known to be conserved in yeast telomerase RNA besides canonical Sm site. Mutagenesis confirmed that Sm-proteins were not involved in the protection of hpTER from RNAse degradation for the formation of precise 3’-end after the initial transcript cleavage due to the first splicing event. Hp telomerase possesses the unique feature, it is able to add additional dT nucleotide which is not expected according to the telomere sequence. Analysis of telomere ends allowed us to prove that this nucleotide is a last nucleotide at telomere in 90% of cases. The mutation of this position at hpTER lad to a dramatic influence on telomere length thus providing an evidence that H. polymorpha telomerase utilizes reverse transcription of nontelomeric nucleotide to control telomere elongation.