Telomeres are specialized heterochromatic DNA–protein complexes at the ends of linear eukaryotic chromosomes. As in centromeres, the nucleosomes around which telomeric DNA is coiled contain modified histones that promote the formation of constitutive heterochromatin.
Telomere structure, function, and evolution
Telomeric DNA sequences are almost always composed of moderately long arrays of short tandem repeats that, unlike centromeric DNA, have generally been well conserved during evolution. In all vertebrates that have been examined, the repeating sequence is the hexanucleotide TTAGGG (Table1). The repeats are G-rich on one of the DNA strands (the G-strand) and C-rich on the complementary strand. On the centromeric side of the human telomeric TTAGGG repeats are a further 100–300 kb of telomere-associated repeat sequences (Figure 1A). These have not been conserved during evolution, and their function is not yet understood.
Table1. EVOLUTIONARY CONSERVATION OF TELOMERIC REPEAT SEQUENCES IN EUKARYOTIC CELLS
Fig1. At telomeres, highly conserved oligonucleotide repeats are bound by specialized proteins to form a protective loop. (A) Telomere structure. The DNA at the very ends of human chromosomes is defined by a tandem array of roughly 1700–2500 copies of the hexanucleotide TTAGGG (that is conserved in vertebrates, see Table 1). The G-rich strand, however, protrudes at the terminus to form a single-stranded region composed of ~30 TTAGGG repeats. The array of distinctive, conserved short repeats is bound by the shelterin (or telosome) complex (not shown for simplicity; two of its subunits, the telomere repeat binding factors TRF1 and TRF2, directly bind to double-stranded regions, while POT1 can bind to the single-stranded repeats). Like centromeric DNA, telomeric DNA has modified histones that act as signals for forming constitutive heterochromatin. (B) T-loop formation. The single-stranded terminus of the G-rich strand can loop back and invade the double-stranded region by base-pairing with the complementary C-rich strand sequence. The resulting T-loop is thought to protect the telomere DNA from natural cellular mechanisms that repair double-strand DNA breaks. (C) Electron micrograph showing T-loop formation. The example shows formation of a ~15 kb T-loop at the end of an interphase human chromosome (after fixing, deproteination, and artificial thickening to assist viewing). (From Griffith JD et al. [1999] Cell 97:503–514; PMID 10338214. With permission from Elsevier.)
The (TTAGGG)n array of a human telomere often spans about 10–15 kb (see Figure 1A). A very large protein complex (called shelterin, or the telosome) contains several components that recognize and bind to telomeric DNA. Of these components, two telomere repeat binding factors (TRF1 and TRF2) bind to double-stranded TTAGGG sequences.
As a result of natural difficulty in replicating the lagging DNA strand at the extreme end of a telomere, the G-rich strand has a single-stranded overhang at its 3′ end that is typically 150–200 nucleotides long (see Figure 1A). This can fold back and form base pairs with the other, C-rich, strand to form a telomeric loop known as the T-loop (Figure 1B and C).
The T-loop probably represents a conserved mechanism for protecting chromosome ends. If a telomere is lost following chromosome breakage, the resulting chromo some end is unstable; it tends to fuse with the ends of other broken chromosomes, or to be involved in recombination events, or to be degraded. Telomere-binding proteins, notably the telosome component POT1, binds to single-stranded TTAGGG repeats and can protect the terminal DNA in vitro and perhaps also in vivo.
Telomerase and the chromosome end-replication problem
During DNA synthesis, the DNA polymerase extends the growing DNA chains in the 5′ → 3′ direction. One of the new DNA strands, the leading strand, grows in the 5′ → 3′ direction of DNA synthesis but the other strand, the lagging strand, is synthesized in pieces (Okazaki fragments) because it must grow in a direction opposite to that of the 5′ → 3′ direction of DNA synthesis. A succession of “backstitching” syntheses is required to produce a series of DNA fragments whose ends are then sealed by DNA ligase (Figure 2).
Fig2. The problem with replicating the extreme ends of DNA in linear chromosomes. In normal DNA replication by DNA-dependent DNA polymerases, an existing DNA strand is used as a template for making a complementary new DNA strand. Here, as the replication fork advances in the upward direction, it can synthesize a continuous DNA strand, the leading strand, upward in the 5′ → 3′ direction from one original DNA strand (colored purple), but for the pale blue original strand, the 5′ → 3′ direction for DNA synthesis is in a direction opposite to the upward direction of the replication fork. Here, the lagging strand is synthesized in short pieces, called Okazaki fragments, starting from a position beyond the last fragment and moving backward toward it. (DNA-dependent DNA polymerases use short RNA primers to initiate the synthesis of DNA; the RNA primers are degraded, DNA synthesis fills in, and adjacent Okazaki fragments are ligated.) The question mark indicates a problem that is reached at the very end of the strand: how is synthesis to be completed when there can be no DNA template beyond the 3′ terminus?
Unlike RNA polymerases, DNA polymerases absolutely require a free 3′ hydroxyl group from a double-stranded nucleic acid from which to extend synthesis. This is achieved by employing an RNA polymerase to synthesize a complementary RNA primer that primes synthesis of each of the DNA fragments used to make the lagging strand. In these cases, the RNA primer requires the presence of some DNA ahead of the sequence to be copied that serves as its template. However, at the extreme end of a linear DNA molecule, there can never be a template ahead of the sequence to be copied, and a different mechanism is required to solve the problem of completing replication at the ends of a linear DNA molecule.
A solution to the end-replication problem is provided by a specialized reverse transcriptase (RNA-dependent DNA polymerase) that completes leading-strand synthesis. Telomerase is a ribonucleoprotein enzyme whose polymerase function is critically dependent on an RNA subunit, TERC (telomerase RNA component), and a protein subunit, TERT (telomerase reverse trancriptase). At the 5′ end of vertebrate TERC RNA is a hexanucleotide sequence that is complementary to the telomere repeat sequence (Figure 3). It will act as a template to prime extended DNA synthesis of telomeric DNA sequences on the leading strand. Further extension of the leading strand provides the necessary template for DNA polymerase to complete synthesis of the lagging strand.
Fig3. Telomerase uses a reverse transcriptase and a noncoding RNA template to make new telomere DNA repeats. The telomerase reverse transcriptase (TERT) is an RNA-dependent DNA polymerase: it uses an RNA template provided by its other subunit, TERC (telomerase RNA component). Only a small part of the RNA is used as a template—the hexanucleotide that is shaded—and so the telomeric DNA is extended by one hexanucleotide repeat (blue shading). Repositioning of the telomeric DNA relative to the RNA template allows the synthesis of tandem complementary copies of the hexanucleotide sequence in the RNA template.
In humans, telomere length is known to be highly variable and telomerase activity is largely absent from adult cells except for certain cells in highly-proliferative tissues such as the germ line, blood, skin, and intestine. In cells that lack telomerase, the extreme ends of telomeric DNA do not get replicated at S phase and their telomeres progressively shorten. Telomere shortening is effectively a way of counting cell divisions and has been related to cell senescence and aging. Cancer cells find ways of activating telomerase, leading to uncontrolled replication.
