A fundamental difference between eukaryotic and bacterial replication arises because eukaryotic chromosomes are linear and thus have ends. The 3′-OH group needed for replication by DNA polymerases is provided at the initiation of replication by RNA primers that are synthesized by primase, as stated earlier. This solution is temporary because, eventually, the primers must be removed and replaced by DNA nucleotides. In a circular DNA molecule, elongation around the circle eventually provides a 3′-OH group immediately in front of the primer (Figure 9.14a). After the primer has been removed, the replacement DNA nucleotides can be added to this 3′-OH group. But what happens when a DNA molecule is not circular, but linear?

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THE END-REPLICATION PROBLEM In linear chromosomes with multiple origins, the elongation of DNA in adjacent replicons provides a 3′-OH group preceding each primer (Figure 9.14b). At the very end of a linear chromosome, however, there is no adjacent stretch of replicated DNA to provide this crucial 3′-OH group. When the primer at the end of the chromosome has been removed, it cannot be replaced by DNA nucleotides, so its removal produces a gap at the end of the chromosome, suggesting that the chromosome should become progressively shorter with each round of replication. Chromosome shortening would mean that when an organism reproduced, it would pass on shorter chromosomes than it inherited. Chromosomes would become shorter with each new generation and would eventually destabilize. This inability of the processes we have discussed to replicate the ends of linear chromosomes has been termed the end-replication problem. Chromosome shortening does in fact take place in many somatic cells, but in single-celled eukaryotes, germ cells, and early embryonic cells, chromosomes do not shorten and self-destruct. So how are the ends of linear chromosomes replicated?

TELOMERES AND TELOMERASE The ends of eukaryotic chromosomes—the telomeres—possess several unique features, one of which is the presence of many copies of a short repeated sequence. In the protozoan Tetrahymena (in which these repeated sequences were first discovered), this telomeric repeat is TTGGGG, and this G-rich strand typically protrudes beyond the complementary C-rich strand (Figure 9.15a; see also the section Telomere Structure in Chapter 8):

The single-stranded protruding end of the telomere, known as the G overhang, can be extended by telomerase, an enzyme with both a protein and an RNA component (also known as a ribonucleoprotein). The RNA part of the enzyme contains from 15 to 22 nucleotides that are complementary to the sequence on the G-rich strand. This RNA sequence pairs with the overhanging 3′ end of the DNA (Figure 9.15b) and provides a template for the synthesis of additional DNA copies of the repeats. DNA nucleotides are added to the 3′ end of the G overhang one at a time (Figure 9.15c); after several nucleotides have been added, the RNA template moves down the DNA and more nucleotides are added to the 3′ end (Figure 9.15d). Usually, from 14 to 16 nucleotides are added to the 3′ end of the G-rich strand.

In this way, the telomerase can extend the 3′ end of the chromosome without the use of a complementary DNA template (Figure 9.15e). How the complementary C-rich strand is synthesized (Figure 9.15f) is not clear. It may be synthesized by conventional replication, with DNA polymerase α synthesizing an RNA primer on the 5′ end of the extended (G-rich) template. The removal of this primer once again leaves a gap at the 5′ end of the chromosome, but this gap does not matter, because the end of the chromosome is extended at each replication by telomerase, so the chromosome does not become shorter overall.

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Telomerase is present in single-celled eukaryotes, germ cells, early embryonic cells, and certain proliferative somatic cells (such as bone-marrow cells and cells lining the intestine), all of which must undergo continuous cell division. Most somatic cells have little or no telomerase activity, and chromosomes in these cells progressively shorten with each cell division. These cells are capable of only a limited number of divisions; when the telomeres have shortened beyond a critical point, the chromosomes become unstable, have a tendency to undergo rearrangements, and are degraded. These events lead to cell death.

TELOMERASE, AGING, AND DISEASE The shortening of telomeres may contribute to the process of aging. The telomeres of genetically engineered mice that lack a functional telomerase gene (and therefore do not express telomerase in somatic or germ cells) undergo progressive shortening in successive generations. After several generations, these mice show some signs of premature aging, such as graying, hair loss, and delayed wound healing. Through genetic engineering, it is also possible to create somatic cells that express telomerase. In these cells, telomeres do not shorten, cell aging is inhibited, and the cells will divide indefinitely.

Some of the strongest evidence that telomere length is related to aging comes from studies of telomeres in birds. In 2012, scientists in the United Kingdom measured telomere length in red blood cells taken from 99 zebra finches at various times during their lives. The scientists found a strong correlation between telomere length and longevity; birds with longer telomeres lived longer than birds with short telomeres. The strongest predictor of life span was telomere length early in life, at 25 days, which is roughly equivalent to human adolescence. Although these observations suggest that telomere length is associated with aging in some animals, the precise role of telomeres in human aging remains uncertain.

Some diseases are associated with abnormalities of telomere replication. People with Werner syndrome, an autosomal recessive disease, show signs of premature aging that begins in adolescence or early adulthood, including wrinkled skin, graying of the hair, baldness, cataracts, and muscle atrophy. They often develop cancer, osteoporosis, heart and artery disease, and other ailments typically associated with aging. The causative gene, called WRN, has been mapped to human chromosome 8 and normally encodes a RecQ helicase enzyme, which is necessary for the efficient replication of telomeres. In people with Werner syndrome, this enzyme is defective, and consequently, the telomeres shorten prematurely.

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Telomerase also appears to play a role in cancer. Cancer tumor cells have the capacity to divide indefinitely, and telomerase is expressed in 90% of all cancers. As we will see in Chapter 16, cancer is a complex, multistep process that usually requires mutations in at least several genes. Telomerase activation alone does not lead to cancerous growth in most cells, but it does appear to be required, along with other mutations, for cancer to develop. Some experimental anticancer drugs work by inhibiting the action of telomerase.

One of the difficulties in studying the effect of telomere shortening on the aging process is that the expression of telomerase in somatic cells also promotes cancer. To circumvent this problem, Antonia Tomas-Loba and her colleagues created genetically engineered mice that expressed telomerase and carried genes that made them resistant to cancer. These mice had longer telomeres, lived longer, and exhibited fewer age-related changes, such as skin alterations, decreases in neuromuscular coordination, and degenerative diseases. These results support the idea that telomere shortening contributes to aging. TRY PROBLEM 28