To Study the Role of Telomerase in Cellular Replication

An introduction to telomeres:

A telomere is an important structure of the chromosome. Every chromosome consists of a special DNA-protein complex with a particular sequence. Thus telomeres are the caps at the end of the chromosomes. A human telomere is 3 to 20 kilobases in length. It consists of tandem repeats of TTAGGG-3’ sequence. The telomeric sequence is 3000 times repeated. When you go out on a sunny day, would you not prefer to wear a cap? Sun’s scorching heat would be unbearable and wearing a cap would protect you from the damaging heat. Similarly, telomeres are caps on the chromosomes.

 There are three important functions of telomeres:

1.     Telomeres provide structural stability to the chromosomes by sealing their ends.

2.     They protect the ends of the chromosomes from damage or formation of rings.

3.     Telomeres protect the chromosomes from fusing with other DNA.

A telomerase enzyme is a ribonucleoprotein complex. It acts like a reverse transcriptase. It plays a crucial role in terminal chromosomal maintenance. Telomerase adds telomeric repeats to the chromosomal ends or DNA termini. A telomerase enzyme compensates for incomplete replication. Telomerase can elongate the telomeres. They enable the cells to distinguish between natural chromosomal ends and double-stranded breaks. They maintain chromosome stability.

Species	Telomere repeat sequence	Telomerase RNA template sequence
Human	5’-TTAGGG-3’	5’-CUAACCCUAAC-3’
Oxytricha	5’-TTTTGGGG-3’	5’-CAAAACCCCAAAACC-3’
Tetrahymena	5’-TTGGGG-3’	5’-CAACCCCAA-3’

Table: The telomeric repeat sequences and telomerase RNA template sequences in humans, oxytricha, and tetrahymena respectively.

Structure of a telomere:

A telomere consists of the following components:

1.     DNA sequence:

A telomere consists of 5’TTAGGG 3’ sequence. A hexamer unit is present in 2000 copies.

2.     Loops

Several studies indicate the occurrence of telomeres in the form of loops. Most of the telomeres end in a loop known as T-loop, where the double-stranded telomeric tract curves around. The T-loop formation protects from exonucleases. There is a displacement loop of TTAGGG repeats known as a D loop. Telomeres can also form specialized structures known as G-quadruplex DNA. These structures are composed of guanine tetrads or G-quartets. Guanine tetrads are square planar arrays of four, hydrogen-bonded guanines. Hoogsteen base pairing is common among them. This G-quartets stack upon each other and provide telomere protection.

3.     Protein components

·        TRF1: It is known as a telomere repeat binding factor 1. It binds to the telomere at T-loop. TRF-1 inhibits telomerase-dependent elongation.

·        TRF 2: It is known as telomeric repeat binding factor 2. It is involved in the formation of T-loop. The overexpression of TRF2 in somatic cells leads to telomere shortening.

·    hRAP 1: It is a human homolog of yeast protein. It is involved in determining the length of the telomere.

·    TIN 2: It is known as a TRF-1 interacting nuclear factor. It promotes pairing of telomere repeats.

·        TANK1: It promotes telomere elongation.

Image 1: Chromosomes showing their telomeres

What is telomere shortening?

DNA replication is an important process of the cell. In this process, DNA is duplicated using replication enzymes. However, during DNA replication, the enzymes may skip replicating the ends of the DNA. As a result, few telomeric sequences get skipped. Thus, the replicated DNA is slightly shorter than the original. After many replications, the telomere sequences become too short. As a result, the cell division stops and the cell undergo apoptosis. This phenomenon leads to aging in human. The process of telomere shortening is observed in somatic cells and not in the germ cells, antibody-producing cells, and cells which constantly replace the gut epithelium. The telomerase enzyme is prominently present in these cells. That is why these cells do not undergo telomere shortening. Somatic cells are deficient in the telomerase enzyme.

There are two ways in which the telomere shortening occurs:

The extreme 3’ end of the DNA is difficult to copy. The natural position of the priming site may be beyond the end of the template. Thus, the lagging strand copy is incomplete, because the last Okazaki fragment is not complete. The resulting daughter molecule has a 3’ overhang and gives rise to a grand-daughter molecule which is shorter than the original one.

Another reason for a shortened telomere is the position of the primer. It is at an extreme 3’ end of the lagging strand.

The length of the telomere is a useful parameter in the process of aging.

What is telomere extension?

The telomerase contains a unique RNA-protein complex. The 5’ end of the telomerase consists of 5’-CUAACCCUAAC-3’ sequence. The central region of this sequence is a reverse complement of the telomere repeat. This repeat has a 5’- TTAGGG-3’ sequence.

The extension of telomeric DNA follows five steps. First, the telomerase RNA pairs with the ends of the molecule. Next, the telomeric DNA gets extended at a short distance. A stem-loop structure determines the length of the telomeric DNA. After extension of the telomeric DNA, the telomerase moves further through the translocation process. It starts base pairing the next fragment. In this way, the telomere gets extended.

The completion of telomere extension is unique. A new Okazaki fragment is primed and synthesized, which converts 3’ extensions into the complementary double-stranded ends. The t-loop is formed when a free 3’ end of telomere loops back and invades the double helix.

A process of replicative senescence takes place in the cells. The changes in the structure of the telomeres cause replicative senescence. Cellular senescence leads to changes in cell morphology and gene expression. It is triggered when cells acquire few critically short telomeres.

Eventually, the telomeres become very short. The telomere-protein complex gets disrupted and leads to DNA damage. P53 involved apoptosis is a similar kind of damage. Thus, for a normal senescent, further cell division is blocked.

The finite ability of telomeric DNA replication is known as the Hayflick limit.

Image 2: Telomere extension

Telomeres and cancer:

Most of the cancer cells have active telomerase enzyme. As a result, these cells keep on multiplying. The remaining cells employ an important mechanism known as alternative lengthening of telomeres (ALT) for telomere maintenance.

Most of the cancer cells contain telomerase, a crucial factor for the immortality of the cells.

Suppose there is a mutation in a gene controlling normal cell cycle arrest. Such a cell will divide in spite of having a very short telomere. This cell has the capability of becoming immortal. It may also carry plenty of mutations triggering to cancer.

“In sum, the primary cause of the cancer is a cellular mutation. Telomerase activity and telomere extension are secondary to cancer.”

What are anti-telomerase drugs?

Anti-telomerase drugs work against the telomerase. These drugs target the action of telomerase and block this enzyme. As a result, the cells do not undergo division. However, some side effects may occur due to the complete blocking of telomerase. Cells such as antibody-producing cells, the immune cells, germ cells, and other cells are dependent on telomerase. Thus there could arise some serious effects of using anti-telomerase drugs.

Conclusion:

More research has to be carried out before launching anti-telomerase drugs. Also, we must note that telomerase activity is not the only cause of cancer. Smoking, consumption of alcohol, pollution, chemical mutagens, and other factors may also contribute to the risk of cancer. Mainly these factors induce mutations in the cells. Thus, for designing a cancer drug, the focus should be on the underlying mutations.

References:

[1] Telomeres and Telomerase in Aging, Disease, and Cancer: Molecular Mechanisms, K. Lenhard Rudolph

[2] Genome Instability in Cancer Development, Erich A. Nigg

[3] Topics in Anti-Cancer Research, Atta-ur Rahman, Khurshid Zaman

[4] Concepts Of Genetics, 7/E (With Cd), Klug
[5] Molecular Biology of the Gene, 5th Ed, Pearson Education, 2004: Gene, Pearson Education, Inc

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