Telomeres in most organisms are maintained by a specialized reverse transcriptase (RT) known as telomerase, which uses an integral RNA component as the template for the synthesis of one strand of the telomere terminal repeats. The absence of telomerase in normal somatic cells causes chromosomes to shorten progressively with each cell division, leading eventually to inviability. Activation of telomerase in germ cells and cancer cells, however, results in restoration of telomere length and enables these cells to divide indefinitely. The ability to manipulate telomerase activity may therefore lead to new therapeutic strategies for the treatment of cancer and aging.

Our laboratory studies the mechanisms and regulations of telomerase with the ultimate aim of exploiting the medical potential of this enzyme. Several ongoing projects are as follows:

1.Structure/Function analysis of TERT, the catalytic protein component

A crucial component of the telomerase complex is a protein with reverse transcriptase-like features, generically known as TERT (Telomerase Reverse Transcriptase). Alignment of TERTs from diverse organisms revealed 7 highly conserved motifs that are shared with other classes of reverse transcriptases (e.g., HIV reverse transcriptase). These reverse transcriptase motifs are located in the C-terminal region of the polypeptides. In addition to the reverse transcriptase (RT) motifs, TERT can be shown through sequence alignment to possess several N-terminal, telomerase-specific motifs (named "GQ", "CP", "QFP", and "T"), as well as a conserved C-terminal extension (CTE). Our general working hypothesis is that the RT motifs mediate aspects of telomerase enzymology that are in common with other RTs, whereas the telomerase-specific motifs have evolved to perform functions that are unique to telomerase. This notion has by-and-large been confirmed by our structure-function studies. For example, two unique features of telomerase are (1) its stable association with a specific RNA molecule; and (2) its ability to repetitively copy a short RNA template to generate long stretches of telomeric repeats (aka repeat addition processivity). Both features have been shown to require telomerase-specific motifs. A major current interest is in understanding the structural and mechanistic basis of the "GQ" domain, which functions as an "anchor site" that allows telomerase to bind stably to telomeres and add multiple copies of the telomere repeats.

2. Analysis of regulatory components of the telomerase complex.

Analysis of the telomerase complex from the budding yeast Saccharomyces cerevisiae has revealed the existence of two regulatory components (named Est1p and Est3p) that do not affect the association of the core components, but are required for telomere extension in vivo. To gain broader understanding of the function and evolutionary conservation of these non-catalytic components, we analyzed the genome of the pathogenic yeast Candida albicans, and identified reasonable homologues for both regulatory subunits. Disruption of the EST1 and EST3 homologues in Candida caused telomere dysfunction, suggesting that they are indeed orthologues. Interestingly, extracts from the Candida est1 and est3 null strains have a selective defect in extending DNA oligonucleotides in vitro. Based on analysis using an extensive series of primers, we conclude that Candida Est1p and Est3p augments the ability of telomerase to reverse transcribe through selected barriers in the telomere repeat. More recently, we uncovered surprising structural and functional similarities between Est3 and the mammalian telomeric protein TPP1. This finding provides considerable insights on telomerase regulation and evolution. In addition, we are developing new affinity purification strategies for Candida telomerase in order to achieve a complete inventory of telomerase-associated proteins. The inventory will in turn provide the basis for a more comprehensive understanding of telomerase regulation.

3. A novel function of telomerase in telomere protection.

Telomerase has long been known as the key player in telomere maintenance. However, recent studies have pointed to additional novel functions for the telomerase enzyme. In the course of analyzing Candida telomerase mutants, we found unexpectedly that deletion of either the TERT or RNA gene resulted apparent "de-protection" of telomeric DNA such that one of the telomere strands undergoes extensive degradation. Therefore, at least in Candida, telomerase serves a physically protective function at telomeres. The mechanism underlying this protective activity of telomerase is being investigated.

4. Modulation of the template requirement of telomerase by divalent cations.

Telomerase is generally thought to be an obligate RNP, with both the RNA and protein components being required for catalysis. However, we showed recently that in the presence of adequate concentrations of manganese, telomerase can switch to a template- and RNA-independent mode of DNA synthesis, acting in effect, as a Terminal Transferase. This result suggests that the RNA component does not have an obligatory function in the covalent chemistry of the telomerase reaction. Rather, its function must be to modulate protein conformation or contribute to substrate recognition. We are currently examining the potential physiologic relevance of the terminal transferase activity. We are also using the terminal transferase assay to investigate the sequence- recognition properties of telomerase protein(s).