RNA-Dependent Synthesis of RNA and DNA:- Telomerase Is a Specialized Reverse Transcriptase
Telomeres, the structures at the ends of linear eukaryotic chromosomes, generally consist of many tandem copies of a short oligonucleotide sequence. This sequence usually has the form TxGy in one strand and CyAx in the complementary strand, where x and y are typically in the range of 1 to 4 . Telo meres vary in length from a few dozen base pairs in some ciliated protozoans to tens of thousands of base pairs in mammals. The TG strand is longer than its complement, leaving a region of single-stranded DNA of up to a few hundred nucleotides at the 3end. The ends of a linear chromosome are not readily replicated by cellular DNA polymerases. DNA replication requires a template and primer, and beyond the end of a linear DNA molecule no template is available for the pairing of an RNA primer. Without a special mechanism for replicating the ends, chromosomes would be shortened somewhat in each cell generation. The enzyme telomerase solves this problem by adding telomeres to chromosome ends.
Although the existence of this enzyme may not be surprising, the mechanism by which it acts is remark able and unprecedented. Telomerase, like some other enzymes described in this chapter, contains both RNA and protein components. The RNA component is about 150 nucleotides long and contains about 1.5 copies of the appropriate CyAx telomere repeat. This region of the RNA acts as a template for synthesis of the TxGy strand of the telomere. Telomerase thereby acts as a cellular reverse transcriptase that provides the active site for RNA-dependent DNA synthesis. Unlike retroviral re verse transcriptases, telomerase copies only a small segment of RNA that it carries within itself. Telomere synthesis requires the 3end of a chromosome as primer and proceeds in the usual 5→3 direction. Having synthesized one copy of the repeat, the enzyme repositions to resume extension of the telomere (Fig. 1a). After extension of the TxGy strand by telomerase, the complementary CyAx strand is synthesized by cellular DNA polymerases, starting with an RNA primer . The single-stranded region is protected by specific binding proteins in many lower eukaryotes, especially those species with telomeres of less than a few hundred base pairs. In higher eukaryotes (in cluding mammals) with telomeres many thousands of base pairs long, the single-stranded end is sequestered in a specialized structure called a T loop. The single stranded end is folded back and paired with its complement in the double-stranded portion of the telomere. The formation of a T loop involves invasion of the 3end of the telomere’s single strand into the duplex DNA, perhaps by a mechanism similar to the initiation of homologous genetic recombination. In mammals, the looped DNA is bound by two proteins, TRF1 and TRF2, with the latter protein involved in formation of the T loop. T loops protect the 3 ends of chromo somes, making them inaccessible to nucleases and the enzymes that repair double-strand breaks (Fig. 1b). In protozoans (such as Tetrahymena), loss of telomerase activity results in a gradual shortening of telomeres with each cell division, ultimately leading to the death of the cell line. A similar link between telomere length and cell senescence (cessation of cell division) has been observed in humans. In germ-line cells, which contain telomerase activity, telomere lengths are maintained; in somatic cells, which lack telomerase, they are not. There is a linear, inverse relationship between the length of telomeres in cultured fibroblasts and the age of the individual from whom the fibroblasts were taken: telomeres in human somatic cells gradually shorten as an individual ages. If the telomerase reverse transcriptase is introduced into human somatic cells in vitro, telomerase activity is restored and the cellular life span increases markedly. Is the gradual shortening of telomeres a key to the aging process? Is our natural life span determined by the length of the telomeres we are born with? Further research in this area should yield some fascinating insights.
FIGURE 1 The TG strand and T loop of telomeres. The internal template RNA of telomerase binds to and base-pairs with the DNA’s TG primer (TxGy). 1 Telomerase adds more T and G residues to the TG primer, then 2 repositions the internal template RNA to allow 3 the addition of more T and G residues. The complementary strand is synthesized by cellular DNA polymerases (not shown). (b) Proposed structure of T loops in telomeres. The single-stranded tail synthesized by telomerase is folded back and paired with its complement in the duplex portion of the telomere. The telomere is bound by several telomere-binding proteins, including TRF1 and TRF2 (telomere repeat binding factors). (c) Electron micrograph of a T loop at the end of a chromosome isolated from a mouse hepatocyte. The bar at the bot tom of the micrograph represents a length of 5,000 bp.
