Molecular genetics of telomeres and telomerase
Telomeres are the terminal protein-DNA complexes of linear eukaryotic chromosomes, and are essential to ensure chromosome integrity and stability. Broken chromosome ends, lacking telomeres, show a propensity to fuse with each other and are also susceptible to degradation by exonucleases. Among a wide variety of eukaryotic species, the telomeric DNA consists of typically G-rich tandem repeats, 5-8 bp in length. These repeats are synthesized by telomerase, a telomere-specific RNP polymerase, which uses an internal RNA moiety as a template sequence for this procedure. In a reverse-transcriptase like manner, telomerase copies part of this RNA sequence into DNA.
The mechanism of telomere elongation by telomerase. In this example the telomerase enzyme is synthesizing the repeated sequence TTGGGG, which is the telomeric sequence of Tetrahymena thermophila.
The first detection of telomerase activity, in the ciliateTetrahymena thermophila, was followed by its detection in a variety of organisms including vertebrates, yeast, and plants. In the absence of telomerase activity, telomeres shorten with each cell division. Normal human somatic cells lack detectable telomerase activity, whereas telomerase is activated in germ cells, immortalized cells and the majority of primary tumors. The correlation between telomerase activity and tumor growth has spurred investigations of the possiblities to use telomerase activity as a target for anticancer drug treatments.
Our identification of much longer telomeric repeat units (16-26 bp) in several yeast species has expanded the previous range of telomeric repeat sequences to include not only more complex sequences, but also ones that are not necessarily G-rich. Despite a marked telomeric sequence diversity, all the yeast species examined show a conserved core. This may be partly explained by the preservation of a binding site for the RAP1 protein.
The length of telomeres are regulated, so that each species has a defined average mean length. The RAP1 protein has been shown to play a key role in a negative-feedback mechanism that controls the length of telomeric repeat tracts in yeast. A model has been proposed where the number of bound RAP1 protein molecules is sensed by the cell, and is used to measure the length of the telomere tract.
The "protein counting" model for telomere length regulation. The RAP1 proteins bind to the telomeric DNA. When a threshold number is reached, further telomere elongation by telomerase is inhibited.
How the "protein counting" mechanism is mediated is still largely unknown, but it has been proposed that the binding of a critical number of RAP1 protein molecules alters the shape of telomeres so that the telomerase enzyme can no longer access the end. When the telomeres shorten and the number of protein molecules decrease, the enzyme would regain its ability to bind and elongate the telomere. Several other yeast telomere binding proteins are involved in the assembly of the functional telomere cap of the yeast chromosome, and are implicated in the regulation of telomere length. How the actions of these proteins are coordinated to maintain telomeres within a defined range has yet to be determined.
The telomeric DNA sequences are bound by a number of different proteins which build up a protective cap on the chromosome. The RAP1 protein has been shown to regulate the length of the telomere, probably by controlling the access of telomerase to the end of the chromosome.
We have analyzed the Rap1p protein counting mechanism in two other yeast species; Saccharomyces castellii and Saccharomyces dairenensis, and have shown that they have RAP1 proteins with homologous functions. These species offer an advantage in the prediction of the number of bound Rap1p molecules, because they have homogeneously repeated telomeric DNA sequences, and thus constitute valuable new models for the analyses of the protein counting mechanism.
Fundamental knowledge of telomere maintenance will be of importance for the establishment of the role of telomerase in tumorigenesis. In our project we are characterizing the telomerase enzyme and the mechanisms of its DNA synthesizing activity, and we want to determine what factors that interact with telomerase to regulate its biochemical activity. We recently isolated the S. castellii CDC13 homolog (scasCDC13) and determined that the full-length protein specifically binds single-stranded telomeric DNA. The minimal binding site is an octamer sequence which overlaps the Rap1 binding site. The four nucleotides of most importance for the sequence specific binding were found to be conserved among telomeric sequences of various different species, including those in human telomeres. Thus, further analysis of scasCdc13p function is promising interesting data on the details of the molecular mechanisms involved in telomere maintenance.