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

© Copyright, 2018  All Rights Reserved

Genetics of Cancer

The incidences of cancer started shooting up in the year 1991. Since a decade the cancer cases increased by a factor of three. There is simply no complete cure to this dreadful condition. It involves changes in the genome leading to uncontrolled cellular proliferation, transformation, invasion, metastasis, apoptosis suppression, and angiogenesis. The environmental factors such as chemicals, radiations, viruses, microbes, and hormones cause cancer. Apart from the above reasons, there are more factors involved. The cell follows a cyclical pattern of division involving different phases. It includes the G1, S, G2, and the M phases respectively. The transition of one phase from the other involves checkpoints. The CDK/cyclin complexes mainly control the cell cycle. The checkpoints determine the damaged DNA. They also help in checking the problems in the cell cycle machinery. Hence, they play a crucial role in permitting normal cells to continue. Problems in the cell cycle checkpoints trigger cancerous conditions. Certain viruses such as retroviruses cause cancer. They increase the oncogenic products. Also, for the normal cells, apoptosis plays a crucial role. The cancer cells do not undergo apoptosis.

Image 1: Cancer cells and normal cells

Cell cycle and cancer:

A cell cycle involves six main checkpoints such as the restriction point, the G1/S DNA damage checkpoint, the S phase DNA damage checkpoint, G2/M checkpoint, centrosome duplication checkpoint, and mitotic checkpoint. The restriction point occurs between the mid to late G1 phase. This point ensures the cell to enter into the S phase after receiving the appropriate signals. The G1/S damage checkpoint occurs at the G1 phase transition. It senses the DNA damage. The S phase DNA damage checkpoint arrests the cell cycle in the later part. It detects the DNA damage or an incomplete replication of DNA. The G2/M checkpoint also detects the damaged DNA. The centrosome duplication checkpoint detects the defects in the centrosome duplication process. This checkpoint also detects centrosome segregation defects. The mitotic checkpoint occurs in the M phase. It checks the formation of mitotic spindles.

The CDK/cyclin complexes control the cell cycle. The cyclin-dependent kinases belong to the class of kinases. The cyclins are known as the regulatory subunits. The CDKs are known as catalytic subunits. The cell cycle checkpoints involve a genetic control. The genes participating in the multiple cell cycle checkpoints are known as gatekeeper genes. These genes prevent the cell cycle progression until the damaged DNA gets repaired.

Phases of the cell cycle	Cyclin-CDK complexes
G1 phase	Cyclin D, CDK-4 Cyclin D, CDK-6
Late G1 phase	Cyclin E, CDK-2
S phase	Cyclin A, CDK-2
G2 phase	Cyclin A, cdc 2
M phase	Cyclin B, cdc 2

Table: All phases of the cell cycle and the associated cyclin-CDK complexes

1.     G1 Phase:

Alterations in the signaling pathways associated with the cyclin-dependent kinases lead to the uncontrolled cell proliferation. Retinoblastoma involves tumor in the retina. It occurs in childhood. The gene responsible for getting mutated and causing the disease is known as the RB gene. It is present on the q arm of the thirteenth chromosome. Deletion or inactivation of both the copies of the RB gene leads to retinoblastoma. The cell loses the protein product pRb.

This phase requires a regulatory protein. It is known as pRb. It gets phosphorylated by cyclin/CDK complex. The pRb binds to the E2F transcription factor and prevents the cell’s entry into the S phase. After the phosphorylation of pRb, it gets inactivated and releases the E2F. Now the cell safely enters the S phase. However, in the retinoblastoma, the cell loses the pRb protein due to RB gene mutation. Hence, the cell enters into the S phase without checking any damaged DNA. Thus, it leads to an unrestrained tumor formation.

2.     G1/S checkpoint:

The tumor suppressor gene known as TP53 gene plays a crucial role in cell cycle arrest and DNA repair. This checkpoint gets invoked due to dsDNA breaks and damage. The product of the TP53 gene is a protein. It is known as p53. It helps in arresting the cell cycle in the G1 phase or the G1/S phase. After the repair of the DNA, the cycle resumes back. However, failure to get repaired leads to apoptosis or cell death. It occurs in the normal cells where p53 gets activated. In the cancer cells, the p53 is not present. Hence, there is no cell cycle arrest and repair of damaged DNA. Thus, the cells form tumors.

3.     G2/M checkpoint:

It is a DNA damage checkpoint. It helps in progressing the cell from the G2 phase to mitosis phase. It maintains the cdc2/ cyclin B1 in an inactive state. The protein p53 also plays a crucial role here.

Image 2: Cell cycle

Cellular proliferation:

Signal transduction involves extracellular growth factors. They regulate cell growth and differentiation. The genes encoding the growth factors or the growth factor receptors may get mutated. Hence, they lead to oncogenic properties. A gene encodes for the signal transducing protein. It is known as ras gene. The transcription factor gets encoded by another gene. It is known as the Myc gene. Mutations in both the genes also cause cancer.

Genes, Viruses, and Cancer:

Cancer involves mutations in three main gene classes. They include proto-oncogenes, tumor suppressor genes, and mutator genes. The products of proto-oncogenes stimulate cell proliferation. The mutant ones are known as oncogenes. They are the active forms of cancer genes. The oncogenes stimulate unregulated cellular proliferation. The RNA viruses also replicate via DNA intermediate. These viruses are known as retroviruses. Upon the retroviral infection, the RNA genome of the viral particle synthesizes a kind of cDNA. It is known as proviral DNA. The viruses also have oncogenes. They are known as viral oncogenes. When they occur in the host cell, these genes are known as cellular oncogenes. The host DNA sequences homologous to that of the virus are known as proto-oncogenes. These genes get activated to oncogenes. Three main methods do this. The first method involves increasing the amount of proto-oncogene product. The second method involves mutations in the coding sequences. Chromosomal translocation also leads to activation of oncogenes.

Apoptosis and Cancer:

The cell death or apoptosis gets triggered in the case of unrepaired damaged DNA or any other unwanted cellular conditions. The failure of the checkpoints in stopping the cell cycle progression also triggers cancer. Cancer also occurs due to the activation of anti-apoptotic genes such as Bcl_2. Thus, many such factors contribute to cancer. 

References:
[1] Human Genetics, 3/e, Gangane
[2] Molecular Genetics of Cancer, John Cowell
[3] The Genetics of Cancer: Genes Associated with Cancer Invasion, Metastasis, Gajanan V. Sherbet, M. S. Lakshmi
[4] API Textbook of Medicine, Ninth Edition, Two Volume Set, Y P Munjal, Surendra K Sharma

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