|Figure 1. Synthesis and maintenance of telomeric DNA by addition of TTAGGG repeat units onto the 3' ends of existing single-stranded telomeric DNA. Telomerase (represented by the "orange" box) contains several protein components (TERT, telomerase-associated proteins) and an RNA subunit (TR).|
Telomeres are specialized nucleoprotein structures that cap the ends of all linear eukaryotic chromosomes. They maintain chromosome stability by protecting ends from exonuclease digestion or illegitimate recombination and also ensure complete chromosome replication and proper segregation.1 Telomeres are composed of telomere-binding proteins and short guanine-rich sequences that are tandomly repeated from less than one hundred to several thousand times. In humans and other vertebrates, the highly evolutionary conserved telomeric DNA is composed of the hexanucleotide repeat TTAGGG.2,3 Two telomere binding proteins have been identified in humans and other mammals that are ubiquitously expressed and present together on most human telomeres throughout the cell cycle. Telomeric repeat binding factor protein (TRF)14-6 and TRF27 are homodimeric proteins that bind to double-stranded telomeric specific repeats. TRF1 and TRF2 share a similar architecture containing an N-terminal dimerization domain and a C-terminal Myb-type DNA binding domain, but differ in the amino acid composition of their N-terminus. TRF1 is a negative regulator of telomere length and may act by inhibiting the action of telomerase.8 Recent evidence implicates TRF2 in maintaining the integrity of chromosome ends by preventing end-to-end fusions.9
Because conventional DNA replication machinery cannot copy extreme terminal sequences of the lagging-strand during replication of linear chromosomes, 50-200 base pairs of telomeric DNA will be lost during each successive cell division.10,11 Eventually, the "end-replication" problem will lead to critically shortened telomeres that can no longer form the cap structure to protect the ends of chromosomes from damage, thereby triggering replicative senescence or cell death. In nearly all eukaryotes, this problem is remedied by producing telomerase, a ribonucleoprotein complex that synthesizes and maintains telomeric DNA by adding the repeat units onto the 3' ends of existing single-stranded telomeric DNA.12,13 Telomerases contain several protein components and a RNA subunit.14-16 Studies of lower eukaryotes suggest that telomerase may function as a dimer or higher order oligomer to perform single or multiple repeat additions.17,18
The catalytic subunit has recently been cloned from a variety of species. It is a specialized reverse transcriptase (RT) that relies on an associated RNA to provide a template for the synthesis of telomeric DNA repeats.19-23 Accordingly, the telomerase catalytic subunit is designated TERT (telomerase reverse transcriptase). TERTs are large proteins (103-134 kDa). All share sequence motifs common to conventional RTs. They also display sequence features unique to telomerase RTs that are essential for telomerase activity. In contrast to conventional RTs, which can copy long stretches of DNA or RNA, the polymerization activity of TERTs is somehow restricted to copying a short sequence of RNA template repeatedly. Human TERT has been previously called hTRT,22 hTCS1,24 TP2,20 and hEST2.23
The intrinsic RNA component, referred to as TR (telomerase RNA), is associated with several protein components of telomerase, including TERT. All TRs contain a sequence that is complementary to one unit of the telomeric repeat.25-27 Over 30 TRs have been cloned varying in size from 159 nucleotides in Tetrahymena to 1300 nucleotides in yeast. Human and mouse TR are about 400 nucleotides in length. TRs display low sequence homology, but share a conserved predicted secondary structure consisting of a stem, a pseudoknot, and a set of stem-loop structures.28
Besides TERT and TR, which are both necessary and sufficient to form the catalytic core of telomerase,29, 30 several proteins that complex with the core enzyme have been identified in a number of diverse species.28, 31 Unlike the TERTs, which are phylogenetically conserved, the telomerase-associated proteins appear to lack structural and primary sequence conservation.28, 31 One telomerase-associated protein, called TP1 in mouse and human32 and TLP1 in rat,33 is a large protein (230-240 kDa) that binds TR. TP1/TLP1 is also a component of vaults, which are ubiquitous cytoplasmic ribonucleoprotein particles of unknown function.34 The function of telomerase-associated proteins is generally not known, but they may act to regulate telomerase activity. In addition to human TP1, chaperones p23 and hsp90 also associate with the human telomerase complex by binding to TERT.35 In vitro and in vivo studies support an essential role for p23 and hsp90 in the assembly of active human telomerase. It is not known if this occurs in other species.
Telomerase and telomeres play important roles in determining cell fates such as replicative senescence (inherent of organismal aging) or unlimited replicative capacity characteristic of maligancies. Most normal human somatic cells lack both detect-able telomerase activity and TERT expression22, 23 and their telomeres continuously shorten with each successive cell division.36, 37 Shortening of telomeres to a critical length may act as a "mitotic clock" triggering cellular senescence.11, 36, 38 Interestingly, TR is expressed in most telomerase negative normal cells.39, 40 Restoration of telomerase activity in these cells through ectopic expression of TERT leads to telomere lengthening with a significant extension of life span.41, 42 Telomerase is constitutively active in germ cells, the majority of primary tumors, and immortalized cells where it stabilizes telomere length.43-46 Low telomerase activity is also found in other normal cells including activated lymphocytes47 and stem cells in renewal tissues such as intestinal crypts and the basal layer of the skin.48, 49 Low telomerase activity provides for an increased proliferative capacity of these tissues, but is insufficient to prevent telomere shortening. In marked contrast to human tissues, significant telomerase activity is present in most normal mouse tissues, suggesting that human and mouse cells fundamentally regulate telomerase expression differently.50
Interest in the potential application of telomerase as a diagnostic and prognostic tumor marker is growing steadily. This stems from the observation that greater than 85% of tumor samples express telomerase activity,51, 52 which suggests a key role for telomerase in sustaining the growth of most tumors. Also important is the development of the highly sensitive and efficient TRAP (Telomeric Repeat Amplification Protocol) assay,43, 53, 54 that can measure telomerase activity in a quantitative and reproducible format. Although promising correlations have been reported, a further understanding of the dynamics of telomerase and tumorigenesis is needed before a clinically useful profile of telomerase can be established for diagnostic and prognostic applications.