ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 11, pp. 1704-1718 © The Author(s) 2023. This article is an open access publication.
Published in Russian in Biokhimiya, 2023, Vol. 88, No. 11, pp. 2066-2083.
1704
REVIEW
Olovnikov, Telomeres, and Telomerase.
Is It Possible to Prolong a Healthy Life?
Yegor E. Yegorov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
e-mail: yegorov58@gmail.com
Received July 20, 2023
Revised August 29, 2023
Accepted August 31, 2023
Abstract— The science of telomeres and telomerase has made tremendous progress in recent decades. In this review, we
consider it first in a historical context (the Carrel–Hayflick–Olovnikov–Blackburn chain of discoveries) and then review
current knowledge on the telomere structure and dynamics in norm and pathology. Central to the review are consequences
of the telomere shortening, including telomere position effects, DNA damage signaling, and increased genetic instability.
Cell senescence and role of telomere length in its development are discussed separately. Therapeutic aspects and risks of
telomere lengthening methods including use of telomerase and other approaches are also discussed.
DOI: 10.1134/S0006297923110032
Keywords: telomeres, telomerase, aging, carcinogenesis, Olovnikov, telomere crisis, cell senescence, inflammatory aging,
genetic instability
HISTORICAL INTRODUCTION
The end regions of chromosomes visible in a light
microscope have been termed by cytologists telomeres.
These are very large structures covering regions of mil-
lions of DNA base pairs. Descriptions of telomeres as
special structures that ensure integrity of chromosomes
first appeared in the Muller’s work performed on the
Drosophila chromosomes in 1938 [1], and in the works
of McClintock performed on the maize chromosomes
in 1938-1941 [2-4]. The essence of these early observa-
tions is that a broken chromosome remains unstable un-
til it acquires a new telomere either by recombination or
de novo. Modern biologists consider the much smaller
end regions of chromosomes, thousands of nucleotide
pairs long, as telomeres.
Although the idea that the ends of linear chromo-
some require special stabilization has been recognized
by the scientific community, it had not been especially
fruitful until it was connected to the cell senescence and
cell immortalization.
Back in the 19th century, after numerous proofs of
cell theory, it became clear that there are mortal and
immortal cells in multicellular organisms, including hu-
mans. Immortal cells must exist because living organ-
isms, being the product of long evolution, are still alive.
At the same time, our cells die, including via pro-
grammed death. Early experiments on the cell cultiva-
tion pointed to the potential immortality of cells. Ex-
periments on serial transplantation of tumor cells in
peritoneal cavities of rats (end of the XIX century) [5],
then the famous experiment of Alexis Carrel, when
chicken cells were continuously multiplied for 30 years,
created a false belief that all cells are immortal [6].
Retrospectively, we realize that experiments demon-
strating cell death in culture are of little informative
value. Such result can always be attributed to a technical
error. A prime example is the hard-won published paper
by Leonard Hayflick, who claimed that “in our hands,
cells are not capable of infinite division” [7]. Future
Nobel laureate Peyton Rous wrote just one word as a
review – “nonsense” [8]. Nevertheless, the “Hayflick
limit” turned out to be real, and the term became gen-
erally accepted.
The main reason for persistence of the view that
cultured cells are immortal was Carrel’s reputation.
Author of the idea of making organs from the patient
cells to avoid rejection (even before the discovery of
blood groups), author of the vascular suture (Nobel Prize
in1912), inventor of masses of devices for transplantation
and sterile work– he was extremely authoritative. Infact,
he developed technology for primary surgical wound
care during World War I, saving thousands of lives.
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BIOCHEMISTRY (Moscow) Vol. 88 No. 11 2023
At the same time, he was the man who proposed “ahu-
mane and economical way of disposing of inferior hu-
man beings in gas chambers” (“should be humanely and
economically disposed of in small euthanasic institutions
supplied with proper gases”), which was realized already
during the Second World War. After 1945 in 20French
cities streets named after Carrel were renamed back.
TheFrench disliked very much those who collaborated
with the Nazis [9-11].
Limited proliferative capacity of cells found in the
Hayflick’s experiments had to be explained. After the
mechanisms of DNA replication became clear in gener-
al terms, a hypothesis on the mechanism of functioning
of a cell division counter was suggested. In 1971, Alexey
Olovnikov proposed the “principle of marginotomy in
template synthesis of polynucleotides”, which claims
that DNA polymerase is unable to fully replicate a lin-
ear template; the replica is always shorter in its initial
part [12]. Gradual shortening of DNA (underreplication)
limits proliferative potential of the cells and can serve as
the basis of the cell division counter in the Hayflick’s
experiments. In the same work it was postulated that
immortal cells should possess an enzyme that completes
chromosome ends.
This enzyme, later called telomerase, was first dis-
covered in 1985 in the protozoan cells [13]. At that time,
the authors thought that the enzyme they found in infu-
soria cells was necessary only for replication of the spe-
cial telomeres of this protozoan. Later they (Nobel lau-
reates Elizabeth Blackburn and Carol Greider) recalled:
“We did not know about Olovnikov’s ideas until 1988,
when Calvin Harley told Greider about them. Intrigued,
Greider, Harley, and their colleagues decided to find
out if the chromosome shortening in human cells occurs
over time.” [14].
The very next year, 1989, telomerase was detected
in human cells and the length of human telomeres was
found to change during development. A year later, telo-
mere shortening during the cell aging was revealed. In
1998, it was proved that telomerase expression induced
by gene insertion leads to cell immortalization.
In addition to Olovnikov’s visionary articles of 1971
and 1973, several of his other important works should
be mentioned. A few months ago, Alexei Matveyevich
passed away, and in writing the historical part of the re-
view, I would like to touch on some of his hypotheses,
both confirmed and not confirmed.
First, few people remember that Olovnikov suggest-
ed that in addition to underreplication, underrepair may
also lead to telomere shortening [15, 16]. Indeed, it was
subsequently found that telomeres shorten at different
rates (per division) depending on the conditions of cell
cultivation [17,18]. Thus, telomere shortening turns not
into a simple division count, but into a total index that
takes into account various factors, including oxidative
stress conditions.
Second, around the millennium boundary,
Olovnikov hypothesized existence of perichromosomal
particles, which are copies of chromosome segments[19].
It was assumed that transcription of these particles yields
some short RNAs controlling many processes related to
spatial and temporal regulation of genes and chromatin
rearrangement. The terms were introduced: redusomes,
chronomers, printomers, fountain RNA, etc. This hy-
pothesis is still waiting for its confirmation. TERRA
(TElomeric Repeat-containing RNAs) could be consid-
ered as distant analogs of such particles (see below).
It should be noted that scientific events related to
the study of cellular immortality have gained wide pub-
licity due to their intensive coverage by the mass me-
dia. The author of this article learned about the noto-
rious 50 divisions that human cells are capable of from
the popular in USSR weekly review of the foreign press
“Za rubezhom (Abroad)”. The term “Hayflick limit”
has entered the encyclopedias. Unlike the Hayflick
and Carrel works, Olovnikov’s work was little known,
also due to the relative isolation of Soviet science from
the world. Only translation of the Olovnikov’s paper
published in Russian in 1971 into English (2 years lat-
er) [20], made it possible to acquaint the world com-
munity with it. And it received a well-deserved recog-
nition 15 years later, leading to an explosive growth in
the number of papers on the role of telomeres in aging
all over the world and, as a result, to the Nobel Prize,
but not to A.M. Olovnikov.
TELOMERS
Telomeres are the ends of chromosomes, and
they must be packaged so that the repair systems do
not confuse them with the double-stranded breaks in
DNA. This is achieved through the ability of telomere
sequences to fold in a special way and through special-
ized proteins that protect these “breaks.” In humans
and all vertebrates, telomeric DNA is represented by the
5′-(TTAGGG)
n
-3′ sequence [21]. At the ends of human
telomeres, there are single-stranded 3′ regions about
100-150 nucleotides long [22] (Fig.1).
This single-stranded region is present both in the
cells with and without telomerase, so it cannot be ex-
plained by telomerase activity. Presence of this free
3′-end is a direct consequence of underreplication of
the end, namely, removal of the 5′-end RNA primer on
the opposite strand and its inability to be filled by DNA
polymerase during replication.
As the structure suggests, this single-stranded site
contains repetitive clusters of three guanines (GGG).
Calculations show that this sequence easily forms non-ca-
nonical structures (triplexes, quadruplexes). G-4 struc-
tures (quadruplexes) can be intramolecular, bimolecular,
and even tetramolecular, i.e., connecting 4DNA strands.
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BIOCHEMISTRY (Moscow) Vol. 88 No. 11 2023
Fig. 1. A simplified scheme of telomere organization.
The strands in them can be parallel and antiparallel. It is
believed that at least 12 guanines are required to form a
quadruplex [23].
There is an alternative, more widely accepted mod-
el of the behavior of the single-stranded 3′-end of DNA.
The results of experiments of De Lange and coworkers
allowed to propose a telomere model based on the telo-
meric loop [24]. According to it, the single-stranded
end together with the proteins interacts with the dou-
ble helix of telomeric DNA. Thus, a telomeric loop is
formed. Length of this loop correlates with the length of
the telomeric repeat measured by independent methods.
Six proteins present in telomeres form a complex
called shelterin. Two proteins (TRF1 and TRF2) bind
the double-stranded regions of telomeric DNA, two
proteins (POT1 and TPP1) bind the single-stranded
DNA, and two more proteins (TIN2 and Pap1) have no
(at least in humans) DNA-binding sites [25, 26] (Fig.1).
Despite the fact that the telomeric regions of DNA
are devoid of protein-coding sequences, they are tran-
scribed. A long noncoding RNA TERRA (TElomeric
Repeat-containing RNA) is formed [27, 28]. TERRA
expression is initiated in subtelomeric regions [29].
Thus, it contains subtelomeric sequences at the 5′-end
and telomeric UUAGGG repeats at the 3′-end [30].
Various events leading to the changes in TERRA ex-
pression are strongly associated with the telomere length
[31, 32]. For example, TERRA expression is highly sen-
sitive to stress [33,34] and depends on epigenetic chang-
es in subtelomeric regions [35]. Interestingly, TERRA is
abundantly detected as part of plasma exosomes and is
able to modulate the inflammatory response [36]. Also,
TERRA expression is altered in Hutchinson–Gilford
progeria [37]. Overall, however, a clear picture of TERRA
function has not yet emerged. It is obvious that TERRA
expression affects numerous heterogeneous cellular
responses, including telomere maintenance, chroma-
tin state, stress response, and inflammation induction.
Involvement of TERRA in carcinogenesis is being inten-
sively studied. Recent articles [38-42] on this subject
can be recommended to the reader.
TELOMERASE
Telomerase is a reverse transcriptase with an in-
tegrated RNA template for telomeric repeat synthe-
sis. The enzyme is based on the protein part of human
telomerase reverse transcriptase (hTERT) and the RNA
component (hTERC). A small part of hTERC contains
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BIOCHEMISTRY (Moscow) Vol. 88 No. 11 2023
Fig. 2. Partial interactome in hTERT regulation (from [49] with permission).
a template for telomeric DNA synthesis [43]. The en-
zyme works as a large complex with molecular mass
of about 0.5 × 10
6
Da. The complex includes hTERT,
hTERC, dyskerin, TCAB1, as well as temporally asso-
ciated proteins pontin, reptin, and chaperones HSP90
and TRiC [44].
As predicted in the Olovnikov’s hypothesis, telo-
merase is expressed in germ, stem, and cancer cells.
Inthe latter case, activation of hTERT, which is prac-
tically absent in normal somatic cells, is observed. In a
relatively small percentage of cases (depending on the
tissue origin of cancer), cancer cells activate an alterna-
tive mechanism of telomere maintenance (ALT) based
on recombination.
The rate-limiting component of telomerase func-
tioning in humans is the hTERT protein. The hTERT
gene has a total length of about 37 kb and consists of
16 exons and 15 introns. In humans, 20 hTERT splicing
variants have been described [45], some of which strong-
ly influence telomerase activity [46]. Splicing of hTERT
changes not only during development, but also during
carcinogenesis [47].
Another level of regulating telomerase activity is
transcriptional. The hTERT promoter contains many
transcription factors binding sites. Among the most
studied regulators are c-Myc, estrogen receptor, HIF-1,
NF-B, Menin, STAT3/5, MAD1, ETS, Sp1/3, USF,
NFX1, etc. [48]. Presence of the sites for numerous ac-
tivators and repressors suggests a very complex system
of gene expression regulation (Fig.2).
In addition to transcription and splicing, regula-
tion of telomerase activity can occur through posttrans-
lational modifications, including phosphorylation [50].
The ability of telomerase to elongate telomeres also de-
pends on many factors, including localization within the
nucleus or in the cytoplasm, and the state of chroma-
tin [51]. Telomerase must undergo a maturation step in
Cajal bodies, where TCAB1 is contained [52,53]. Sup-
pression of activity can be achieved by delaying telomer-
ase in the nucleolus.
Regulation of hTERT is mainly associated with the
cell cycle. It involves cyclin/cyclin-dependent kinase
(cdk) complexes, which regulate transcription factors
that bind to the hTERT promoter. E2F-1 plays a dual
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BIOCHEMISTRY (Moscow) Vol. 88 No. 11 2023
role in the cyclin regulation, acting both as repressor
and as activator. PI3K/Akt, NF-kB, and MAP kinase
cascades activate hTERT. Phosphorylation of the p107
cyclin/cdk complexes as well as binding of estrogen
to its receptor Era relieve the inhibitory effects of the
TGF-b cascade. Through the positive feedback, hTERT
expression activates the PI3K/Akt cascade, which, in
turn, activates the cell cycle via MAD1 and p53 deg-
radation, activates the NF-kB cascade, and blocks the
TGF-β cascade.
EXTRACHROMOSOMAL
FUNCTIONS OF TELOMERASE
Over time, the data on the action of telomerase be-
gan to accumulate, which are difficult to explain only
through its effect on chromosomes. For example, en-
hanced expression of mTERT (murine telomerase) pro-
motes carcinogenesis and wound healing [54]. Enhanced
expression of telomerase alters stem cell functions [55].
A surprising result was obtained when the hTERT gene
was introduced into the cells of a patient suffering from
Niemann-Pick disease, a rare inherited disease char-
acterized by impaired lipid metabolism. Insertion of
hTERT normalized the cell phenotype [56]. Telomerase
has also been shown to affect activity of the glycolysis
genes [57], stimulate transcription of the genes associ-
ated with epithelial-mesenchymal transition (vimentin
and snail1) [58]. Telomerase was shown to influence the
work of NF-kappaB-dependent genes, i.e., participate
in the regulation of inflammation [59]. Mutual effects
of hTERT on the Wnt cascade and vice versa have been
described [60].
Under conditions of oxidative stress, hTERT acts
as a redox regulator, moving into mitochondria, where
it protects mitochondrial DNA and helps maintain the
level of anti-oxidative enzymes [61]. It is not entirely
clear whether hTERT moves from the nucleus to mito-
chondria or whether the newly synthesized protein goes
directly to mitochondria.
Expression of hTERT is involved in epigenetic reg-
ulation by influencing the STAT3 factor, which activates
DNA methyltransferase I [62]. In 2009, it was discovered
that hTERT protein is able to form complex not only
with hTERC, but also with RMRP (RNA component of
mitochondrial RNA-processing endoribonuclease) [63].
Such complex has the RNA-dependent RNA poly-
merase activity, producing long double-stranded RNA.
RMRP mutations are associated with cartilage and hair
hypoplasia disease in humans [64]. It has been suggest-
ed that hTERT may form similar complexes with other
RNAs [65]. We have previously shown that even with-
out RNA, the telomerase protein has non-template DNA
polymerase activity [66]. Subsequently, these data were
confirmed [67].
WHAT HAPPENS
WHEN TELOMERES SHORTEN?
The Olovnikov’s theory of marginotomy suggested
that the trigger for stopping cell divisions and cell death
was impairment of the subtelomeric genes critical for
cell function. This assumption was not explicitly con-
firmed, which was one of the points that prompted him
to create a new hypothesis of aging. Nevertheless, DNA
shortening occurs as a result of end underreplication and
this phenomenon has clear physiological consequences.
What is the mechanism connecting terminal underrep-
lication of chromosomes and cessation of cell growth?
So far, we can clearly see at least three different mecha-
nisms for the effects of telomere shortening on the cells,
which do not agree with each other.
Telomere position effect. In 2001, the effect of
changing expression of the genes located near the telo-
mere when its length changes was first described [68].
As an explanation, the hypothesis of “heterochromatin
stocking” was proposed, which in general can be de-
scribed as follows: telomeric sequences have a special
chromatin packing and special epigenetic changes, and
the effect of this packing extends to nearby genes.
Later, a similar effect was described, but already for
the genes located at a distance [69,70]. A similar expla-
nation has been suggested here: telomeres affect expres-
sion of the genes located in the space next to them as a
result of chromosomal loop formation.
The results of the studies of telomere position effect
are still mostly not very convincing, although a num-
ber of findings are of interest. For example, the hTERT
gene is a subject to the telomere position effect, which
opens up a number of hypothetical mechanisms for how
the telomere length may be maintained in aging and
cancer [71]. At least three mechanisms have been de-
scribed for regulation of the hTERT expression through
formation of chromatin or telomeric loops [72]. Finally,
it should be noted that telomere shortening increases
expression of the ISG15 gene (interferon stimulated
gene 15), which is able to increase inflammation by
stimulating IFNγ production [69]. Thus, there is a di-
rect link between aging and inflammation, which pro-
vides support to the inflammaging theory.
DNA damage signal emergence. After a critical short-
ening of telomeres, a DNA damage signal appears.
Human cells stop proliferation at an average telomere
length of a few thousand nucleotides (i.e., not their
complete shortening) and enter a special state, which is
called cell senescence in English-language literature.
Probably, there is some minimal telomere length
that allows to properly pack the chromosome end so that
it differs from the DNA break. As early as 1997, two years
before the discovery of telomere loops [24], we suggested
a hypothesis about the loop structure of telomeres [73]
(Fig.3).