Abstract
Telomerase is a ribonucleoprotein reverse transcriptase (RT) responsible for the maintenance of one strand of telomere terminal repeats. The catalytic protein subunit of telomerase, known generically as telomerase reverse transcriptase (TERT), exhibits significant homology to RTs encoded by retroviruses and retroelements. The polymerization mechanisms of telomerase may therefore be similar to those of the "conventional" RTs. In this study, we explored the extent of mechanistic conservation by analyzing mutations of conserved residues within the putative "finger" domain of TERT. Previous analysis has implicated this domain of retroviral RTs in nucleotide and RNA binding and in processivity control. Our results demonstrate that residues conserved between TERT and human immunodeficiency virus-1 RT are more likely than TERT-specific residues to be required for enzyme activity. In addition, residues presumed to make direct contact with either the RNA or nucleotide substrate appear to be functionally more important. Furthermore, distinct biochemical defects can be observed for alterations in the putative RNA- and nucleotide-binding TERT residues in a manner that can be rationalized by their postulated mechanisms of action. This study thus supports a high degree of mechanistic conservation between telomerase and retroviral RTs and underscores the roles of distinct aspects of telomerase biochemistry in telomere length maintenance.
Highlights
Telomerase is a ribonucleoprotein that is responsible for maintaining the terminal repeats of telomeres in most organisms [1]
Consistent with a high degree of mechanistic similarity, we show that residues conserved between S. cerevisiae telomerase reverse transcriptase (TERT) and HIV-1 reverse transcriptase (RT) are more likely than TERT-specific residues to be required for enzyme activity
In atomic resolution structural models of HIV-1 RT, motifs 1 and 2 constitute a long -hairpin, the base of which contacts the templating and surrounding bases and the tip of which interacts with the deoxynucleotide triphosphate
Summary
Telomerase is a ribonucleoprotein that is responsible for maintaining the terminal repeats of telomeres in most organisms [1]. The catalytic reverse transcriptase protein subunit TERT, first purified from Euplotes aediculatus as p123, was found to be homologous to Est2p, a yeast protein required for telomere maintenance [7,8,9] Both proteins possess reverse transcriptase (RT)-like motifs, alterations in which can result in inactivation of telomerase activity and reduced telomere length. Mutational analysis of TERT residues equivalent to those located within functional motifs of conventional RTs supports an overall conservation of basic catalytic mechanisms between these two classes of enzymes. Some other crucial RT residues (e.g. Gln in motif BЈ) appear to be less important or even dispensable in telomerase [9] Together, these results suggest that despite the high degree of sequence divergence (Ͻ20% sequence identity), TERT and conventional RTs may possess very similar polymerization mechanisms.
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