PART I

INTRODUCTION
1.1 The Limitation of DNA Replication Process
The DNA replication process involves DNA polymerases, helicase, Single-Strand
Binding Protein (SSBP), and many other factors. While the DNA polymerases
play a big role in creating the new complementary strand of the unwinding DNA,
this enzyme cant fix its own limitation. For linear DNA, such as the DNA in
eukaryotic chromosomes, the fact that a DNA polymerase can only add new
nucleotides at the 3 end of the upstream polynucleotide makes a serious problem.
The replication set gives no chance to add new nucleotides upstream from the 5
end of the new DNA strand (Campbell, et al., 2002).
During DNA replication, synthesis of the lagging strand is discontinuous and
requires the presence of some DNA ahead which is to be served as the template of
an RNA primer. But, at the extreme end of a linear DNA, there can never be such
a template. After the RNA primer is removed, DNA polymerase cant add new
nucleotides in the place where RNA primer formerly located. DNA polymerase
cant undergo new strand synthesis from 3 to 5 (backwards). Because some
nucleotides dont have any pair, they will be degraded. As a consequence,
repetitive replication will make the new DNA molecules shorten gradually. So, a
different mechanism is required to solve the problem of replicating the ends of
linear DNA molecule (Strachan & Read, 1999).
1.2 Telomere and Its Function
Prokaryote organisms have a circular DNA, which makes the DNA have no end
and therefore will not be shorten each time the cell divided. Eukaryote organisms
possess a linear DNA form, by which it have ends that will be shorten each cell
division. Meanwhile, the eukaryote organisms have unique nucleotide sequence at
the ends of chromosomal DNA, called telomere (Campbell, et al., 2002; Anon.,
1998). Telomere doesnt possess any gene; it consists of a long array of repetitive
short nucleotide sequence and some proteins that bound to it. The repetitive

nucelotide sequence may vary between species, for example TTGGGG for
Paramecium, TAGGG for Trypanosoma, TTTAGGG for Arabidopsis, and
TTAGGG for Homo sapiens (Strachan & Read, 1999).
A small fraction of bacterial chromosomes (such as those in Streptomyces sp. and
Borrelia sp.) are linear and possess telomeres too, but the telomeres are very
different from those of the eukaryotic chromosomes in structure and functions.
The known structures of bacterial telomeres take the form of proteins bound to the
ends of linear chromosomes, or hairpin loops of single-stranded DNA at the ends
of the linear chromosomes.
Eukaryotic telomeres normally terminate with 3 single-stranded-DNA overhang
which is essential for telomere maintenance and capping. Telomeres form large
loop structures called telomere loops, or T-loops. Here, the single-stranded DNA
curls around in a long circle stabilized by telomere-binding proteins. At the very
end of the T-loop, the single-stranded telomere DNA is held onto a region of
double-stranded DNA by the telomere strand disrupting the double-helical DNA
and base pairing to one of the two strands (Anon., n.d.).
Telomere acts as a protective cap at the end of DNA sequence. Telomeres appear
to have a role in maintaining the structural integrity of a chromosome. If the
telomeres dont exist or lost, the chromosome ends will be unstable. It may fuse
with the ends from other broken chromosomes, involve in recombination events,
or degraded. The protection against homologous recombination and nonhomologous end joining constitutes the essential capping role of telomeres that
distinguishes them from DNA double-strand breaks. Also, chromosome ends
appear to be tethered to the nuclear membrane, suggesting that telomeres help
position chromosomes and establish the three dimensional architecture of the
nucleus and/or chromosome pairing (Strachan & Read, 1999).
Even a telomere is existing at the end of the DNA string, telomere consist of
nucleotides. Along with the limitation of DNA polymerases, it can be shorten each
time the cell divides. Critical impact will occur if the shortening reaches the gene
of the DNA. Telomere shortening in humans can induce replicative senescence
2

which blocks cell division. This mechanism appears to prevent genomic instability
and development of cancer in human aged cells by limiting the number of cell
divisions (Anon., n.d.). According to this statement, we know that telomere plays
a role to influence the cell aging. In spite of that, after a long time of discovery,
scientists found that there is one important enzyme that possessed by eukaryotes
that can maintain the length of telomeres, called telomerase.
1.3 Telomerase and How It Regulates Telomere
Telomerase was discovered by Carol W. Greider and Elizabeth Blackburn in 1985.
Telomerase, also called telomere terminal transferase, is an enzyme made of
protein and RNA subunits responsible for the maintenance of one strand of the
telomere terminal repeats in most organisms. In other words, this enzyme has the
ability to lengthen telomeres (Chatterjee & Agrawal, 2013).
The canonical function of telomerase requires telomerase reverse transcriptase
enzyme (TERT) which adds telomere nucleotide sequence repeats to the
chromosome ends. Beside TERT, telomerase also needs the telomerase RNA
component (TERC), which provides the template for TERT (Parkinson, et al.,
2008). TERT is the key protein subunit of the telomerase complex, possesses
reverse transcriptase (RT)-like motifs that directly mediate nucleotide addition
(Bosoy, et al., 2002).
Telomerase is a specialized cellular RT. It is a ribonucleoprotein (RNP) complex
and it synthesizes one strand of the telomeric DNAnamely, the strand running
5 to 3 towards the distal end of the chromosomeby copying a short template
sequence within its intrinsic RNA moiety. This action extends the 3 terminal,
single-stranded overhang found at the ends of telomeric DNA. Discovery of this
polymerization action of telomerase established the role of telomerase as a
polymerase that extends one telomeric DNA strand. Synthesis of the
complementary strand of the telomeric repeats is presumed to occur through
lagging strand synthesis by the normal cellular DNA replication machinery. The
resulting array of telomeric DNA repeats attracts and binds a set of DNA
sequence-specific binding proteins. These in turn bind a further set of proteins to
3

build an inferred higher order complex nucleated on the telomeric DNA. Addition
of telomeric DNA onto chromosome ends by telomerase serves to counter balance
the losses predicted from the end-replication problem and from nuclease action at
telomeric DNA ends. Interestingly, although the telomerase mechanism for
telomere maintenance is very widespread among eukaryotes, and seems to have
appeared early in eukaryotic evolution, it is not completely universal. The
telomerase mode of DNA synthesis has some unprecedented properties. Initially,
telomerase RNA was thought to have only a template role. All evidence indicates
that the chemical step of the RT DNA polymerization reaction catalysed by
telomerase is protein-based, mediated by the catalytic aspartate residues in the RT
active site of TERT (Anon., n.d.).
The physiological roles of the proliferation-dependent telomerase regulation in
normal cells remain unclear. During ex vivo expansion of hematopoietic cells,
telomere length decreases despite the presence of telomerase activity, suggesting
that telomerase activity alone is insufficient to completely prevent telomere
shortening in these cells. However, the rate of base pair loss per population
doubling decreases in the presence of telomerase activity during expansion,
indicating that telomerase activation may slow down the rate of telomere erosion
in these cells. Telomerase activation may thus be an adaptive response to protect
excessive telomere loss and possibly may help to extend the proliferative life span
of highly regenerative cells (Bodnar, et al., 1998).
There are several factors and agents that affect telomerase activity, such as:
1. Hormones
Several hormones are involved in telomerase regulation. Regulation by sex
steroid hormones has been extensively analysed. For example, estrogen
activates telomerase in estrogen receptor (ER)-positive cells through the
up-regulation of human TERT (hTERT) mRNA expression.
2. Differentiation inducing agents
Some differentiation-inducing agents can repress telomerase activity in a
variety of cancer cells, probably through indirect action via induction of
cellular differentiation.
3. Growth factors
4

Several growth factors regulate telomerase activity in normal and tumor

cells. Most regulation is achieved by indirect actions, but some growth
factors may work through direct pathways to regulate telomerase.
4. Anti cancer agents

Some anticancer drugs modulate telomerase activity. Cisplatin is a

representative chemotherapeutic agent that cross-links DNA between
guanines. Since telomeres are composed of guanine-rich sequences, it is
possible that cisplatin impairs the telomere structure, leading to the
inhibition of telomerase activity.
5. Histone deacetylase inhibitors
The role of histone deacetylase (HDAC) inhibitors in telomerase
regulation is complex. In a variety of normal cells without telomerase
activity, HDAC inhibitors can induce telomerase activity via upregulation
of hTERT mRNA expression indicating that histone deacetylation is
involved in transcriptional silencing of hTERT in normal cells.
6. Cell cycle regulators
Some cell cycle regulators are involved in telomerase regulation.
Overexpression of p53 efectively represses telomerase activity through
transcriptional down-regulation of hTERT in a variety of cancer cell lines
(Biessman & Mason, 1997).
As telomeres influence cell aging, the presence of telomerase is directly related to
the slowing down of aging. The activity of telomerase can regulate the length of
telomere, therefore the cells life can be maintained and the cell can divide again
and again.

PART 2
CONTENT
2.1 Cell Aging is Related to Telomere and Telomerase Activity
In our life, cell is supposed to grow and divide. This process start from DNA
replication. But cell can only divide until it reaches the limit of replication, called
Hayflick limit. The limit of replication relates with the length of telomeres that
present in the chromosome. A telomere can reach a length of 15.000 base pairs.
However, each time a cell divides, about 25-200 base pairs of telomere are lost
during each division (Aten, 2007). When the telomeres become too short, the
chromosome reaches a "critical length" and can no longer replicate (Blasco, et al.,
1999). Some research explains that the cultures of cells stopped dividing after an
average of 50-70 cumulative population doublings.
Telomerase, as we know, is the enzyme that can lengthen the telomere. However,
telomerase have a very low (almost undetectable) activity in most cells of
multicellular organisms. In most multicellular eukaryotic organisms, telomerase is
active only in germ cells and certain white blood cells. The germ-line cells must
retain an unlimited cell division to allow maintenance of generation after
generation of organisms through evolutionary history. Lymphocytes retain the
ability to divide and differentiate as needed for diverse biological purposes
(Warner & Hodes, 2000).
Because telomerase is not expressed in most cells, the DNA of most somatic cells
in an adult individual is shorter compared to the young one (Campbell, et al.,
2002). There are theories that the steady shortening of telomeres with each
replication in somatic (body) cells may have a role in senescence and in the
prevention of cancer. This is because the telomeres act as a sort of time-delay
"fuse", eventually running out after a certain number of cell divisions and
resulting in the eventual loss of vital genetic information from the cell's
chromosome with future divisions.

As the telomere gets too short, beside stops proliferating, the cell wont work
properly, and more likely to die. When some cells in body are destructed, the
corresponding tissue will get a problem by which it may loss its function
regularly. The non-functioning tissue may affect the anatomical structure and
physiological processes inside the body. As the human reaches an elderly adult
stage, most of cells have damaged and there will be a little/no replication to
conserve it.
In the human body, there is several different celerity of cell division. The fastest
division happens in the skin, thats why the aging of someone can be detected by
seeing the condition of his/her skin, although the accuracy may be questioned.
The medium celerity happens in the liver, skeletal muscle, and adrenal cortex. The
slowest celerity happened in brain, kidney, and heart. Cell aging happen earlier in
man because the telomere lost rate in man is faster than in woman (Bryan, et al.,
1995).
2.2 Telomerase Probably Has an Ability to Reverse Aging

MOUSE
1

MOUSE
2

Figure 2: two mice involved in an experiment on age-related degeneration

The fact that telomerase can reverse the aging process already found by the
researchers after some experiments are done to the mouse (Blasco, et al., 1999).
One of the experiments is done by Mariela Jaskelioff and DePinho (Naik, 2010).
In this experiment, several mice are used as observation objects. Their condition
can represent the condition of 80 year-old humans and were about to die. For the
7

first sample (here represented by mouse 1), they increase the amount of
telomerase in the mouse by devised an estrogen-based drug that would activate
the animals' dormant telomerase gene, known as TERT. The drug, in the form of a
time-release pellet, was inserted under the skin. A month later, the treated mice
showed surprising signs of rejuvenation. The mouse got younger by the
telomerase enzyme. Key organs started to function better. The treated mouse
regained their sense of smell. The male animals' once-depleted testes produced
new sperm cells, dormant brain stem cells, producing new neurons. The spleen,
testes and brain grew in size, and their mates gave birth to larger litters. The
treated animals appear younger than before, though they didn't live longer than
normal mouse. The fur in the treated mice also looks more net and regenerated
than in the mice which are not treated (here represented as mouse 2).
Some chemicals may keep the telomeres long in size, by inducing the expression
of telomerase gene. Therefore, the possibility of the cell to reproduce or divide
again is increase. It means that the aging process can be slow down or reversed.
But in other side, the excess activity of this enzyme can lead to cancer, because
this enzyme may cause the cell to divide continuously and unlimited (Cowell,
1999). Even though the researchers have found that telomerase can reverse aging
in the mouse, further research in human must be done very carefully. The research
may lead to be very complex and requires hard work. But by testing and testing,
researchers believe they can apply it to the human (Mitteldorf, 2013).

PART 3
CLOSING
The DNA replication process, even it is a very complex process, still have a
limitation that inherited from generations to generations. DNA polymerases cant
add new nucleotides from the 5 end of the existing nucleotides. At the extreme
end of a linear DNA, after the RNA primer is removed, there are no nucleotides
ahead to be served as a template. Therefore, DNA polymerase cant add new
nucleotides in the place where RNA primer formerly located, causing some
nucleotides to be degraded. In short, repetitive replication will make the new
DNA molecules shorten gradually (Strachan & Read, 1999).
Eukaryote organisms face this problem by having unique nucleotide sequence at
the ends of DNA, called telomere. Telomeres consist of long repetitive nucleotide
sequence. Telomere doesnt possess any gene, and acts as a cap that protects the
DNA genes from degradation each cell division (Campbell, et al., 2002; and
Anon., 1998). Telomere itselfbecause it is naturally a sequence of nucleotides
also shorten each cell division. If the telomeres have been too short (the genes
nearly exposed), the cell stops dividing and more likely to die. This situation is
called cell aging. If this is happen, the corresponding tissue will be aging too. The
cells are dying and there are a little/no cell division to maintain it. If the tissues are
aging, of course the organism is aging too. Thats why telomere seems to have an
important role in human natural life span (Bryan, et al., 1995).
Scientists have found an enzyme called telomerase that can lengthen the telomere.
Unfortunately, telomerase only activated in several cells, such as germ cells and
lymphocyte cells (Warner & Hodes, 2000). This makes the scientist wonder, if
telomerase activity can be increased in other cells, could it reverse aging?
Experiments done to the mice show that telomerase activity can make old mice
regain its health and maintain its appearance (Naik, 2010). Until now, there are no
experiments done to human to prove that telomerase can slow down the cell aging
or even reverses aging (Mitteldorf, 2013). This fact opens the gate for scientists to

observe this deeper on human, and makes a new hope that molecular biology
using telomerasecan be applied to maintain the healthy life of elderly people.

10

REFERENCES
Anon., 1998. Telomerase Discovery. The Futurist, 32(3), p. 21.
Anon., n.d. Telomere Function. [Online]
Available at: http://www.news-medical.net/health/Telomere-Function.aspx
[Accessed 4 December 2013].
Aten, D., 2007. Cell Biology. s.l.:Biomedical Communications Graduate Program,
Southwestern Medical Program.
Biessman, H. & Mason, J., 1997. Telomere Maintenance Without Telomerase.
Chromosoma, Volume 106, pp. 63-69.
Blasco, M. A., Gasser, S. M. & Lingner, J., 1999. Telomeres and Telomerase.
Genes and Development, Volume 13, pp. 2353-2359.
Bodnar, A., Quelette, M., Frolkis, M. & Holt, S., 1998. Extension of Life-Span by
Introduction of Telomerase into Normal Human Cell. Science, Volume 279, pp.
349-352.
Bosoy, D., Peng, Y., Mian, I. S. & Lue, N. F., 2002. Conserved N-terminal Motifs
of Telomerase Reverse Transcriptase Required for Ribonucleoprotein Assembly In
Vivo. The Journal Of Biological Chemistry, pp. 1-38.
Bryan, T. et al., 1995. Telomere Elongation in Immortal Human Cells Without
Detectable Telomerase Activity. The EMBO Journal, 14(17), p. 42404248.
Campbell, N. A., Reece, J. B. & Mitchell, L. B., 2002. Biologi. 5th ed. Jakarta:
Erlangga.
Chatterjee, S. & Agrawal, S., 2013. Telomerase Reverses Aging. Everyman's
Science, XLVII(6), pp. 359-362.
Cowell, J., 1999. Telomeres and Telomerase in Ageing and Cancer. Age, Volume
22, pp. 59-64.

11

Mitteldorf, J., 2013. Telomere Biology: Cancer Firewall or Aging Clock?.

Biochemistry (Moscow), 78(9), pp. 1054-1060.
Naik, G., 2010. Aging Ills Reversed in Mice. [Online]
Available at:
http://online.wsj.com/news/articles/SB10001424052748703785704575642964209
242180
[Accessed 4 December 2013].
Parkinson, E., Fitchett, C. & Cereser, B., 2008. Dissecting the Non-canonical
Functions of Telomerase. Cytogenetic and Genome Research, Volume 122, p.
273280.
Strachan, T. & Read, A. P., 1999. Human Molecular Genetics. 2nd ed. s.l.:WileyLiss.
Warner, H. R. & Hodes, R. J., 2000. Hype, Hope, and Reality: Telomere Length,
Telomerase, and Aging. Generations, 24(1), pp. 48-53.

12