J. theor. Biol.

(1973) 41, 181-190

A Theory of Marginotomy
The Incomplete Copying of Template Margin in
Enzymic Synthesis of Polym&otides and
Biological Significance of the Phenomenon?
A. M. OLOVNIKOV

Laboratory of Chemistry and Synthesis of Antibodies,

Gamaleya Institute for Epidemiology and Microbiology,
Moscow, U.S.S.R.

(Received 22 June 1972, and in revised form 27 November 1972)

A theory of marginotomy has been proposed to explain the limitation of

the cell doubling potential of clones of normal somatic cells. Marginotomy
of DNA is the shortening of the replica with respect to the template.
Two possible mechanisms of marginotomy are discussed. The first
mechanism is a DNA-polymerase, postulated to have a catalytically
inactive zone lying between the catalytic centre and the outer edge of the
enzyme molecule. The terminal template segment which is equal to the
length of the non-catalytic marginal zone of the DNA-polymerase will
not appear in the replica. The second mechanism of marginotomy results
from a requirement for an initiating RNA-primer for activity of some
DNA-polymerases. In this case the final DNA replica will be shorter than
the template by the length of RNA-primer.
Marginotomy causes the appearance, in the daughters of dividing cells,
of more and more shortened end-genes, the so-called telogenes, with every
new mitosis. The telogenes function as the starting points of end-replicons
in chromosomes and also as “bulIers”, being sacr%ced during successive
mitoses. After the exhaustion of telogenes the cells become aged and are
eliminated due to the loss of some vitally important genes localized in
end-replicons. Marginotomy is therefore responsible for the loss with
age of various cell clones of the body, including some endocrine cell
clones. Therefore marginotomy may be the primary cause of various
disorders of age of the ageing of multicellular organisms.

1. The Problem
This paper considers some aspects of the mechanism of DNA replication
near the ends of polynucleotide chains which may be of considerable
t Preliminary accounts of this work have been published in Dokl. Akad. Nauk SSSR
(1971),201, 1496and reportedat the 9th International Congress of Gerontology in Kiev,
July 2-7 (1972),3,42.
181
182 A. M. OLOVNIKOV

biological interest. I suggest that a special phenomenon exists near the ends
of DNA helix during replication, such that it is not possible for a poly-
nucleotide chain to be completely copied under certain conditions. This
phenomenon, which was not considered in the classical model of DNA
replication (Watson & Crick, 1953) has been called marginotomy (Olovnikov,
1971).
The principle of marginotomy. Marginotomy is the formation of replicas
shortened relative to the template. There are two possible mechanisms of
formation of shortened replicas : the first being “polymerase-dependent”
marginotomy and second “RNA-primer-dependent” marginotomy. Poly-
merase-dependent marginotomy results from the existence of a postulated
catalytically inactive zone (“dead zone”) in the DNA-polymerase molecule.
This zone lies between the catalytic centre of the polymerase, which catalyses
incorporation of successive residues of deoxyribonucleoside-S-3 phosphates
into growing replica, and an edge of the enzyme molecule. This edge is in
contact with the template nucleotide which is furthest from the catalytic
centre. That terminal part of the template, which is equal in length to the
“dead zone” of DNA-polymerase (in Fig. 1, one can see two such zones),
will not be reproduced in the replica. The replica will therefore appear
incomplete, shorter than the template by a length equal to the above-
mentioned inactive zone. Hence, some terminal nucleotides of the template
will remain uncopied in the course of DNA doubling. The second mechanism
of marginotomy is due to the requirements of some DNA-polymerases for
some RNA-primer to initiate DNA replication. This primer must be removed
from the final product and thus the DNA replica will be shortened by the
length of RNA-primer.

2. Description of the Model

(1) Replication of polynucleotide chain of DNA, occurring without
RNA primer, results from the action of one of two essentially different
types of polymerases: (a) monoblock DNA-polymerase (see paragraph 2),
and (b) tandem-DNA-polymerase (see paragraph 6, 7).
(2) A mono-block DNA-polymerase may consist of several subunits, but
acts as a functionally single block with one catalytically active centre.
Functional, the mono-block DNA-polymerase has the following peculiarity:
the polymerase segment, when it protrudes beyond the end of the poly-
nucleotide chain, loses its ability to move along the template (“paralysis of
the segment”), although it retains its catalytic activity. As a result, movement
of the mono-block DNA-polymerase relative to the template stops as soon
as the edge of the enzyme molecule gets beyond the 3’ or 5’ end of the poly-
 MARGINOTOMY THEORY OF AGEING 183
,

FIG. 1. A schematic representation of marginotomic replication (polymerase-dependent

marginotomy). (a) The molecule of single-segment DNA-polymerase. The catalytically
active centre is shown in black. Growth of the replica begins without primer and not
exactly above the 3’end of the template but at the point of localization of the catalytically
active centre of the enzyme. I,,, is the length of marginotomy, i.e. the distance from the
end of the enzyme molecule to its catalytic centre. C, is the length of marginotomy, i.e.
the distance from the catalytic centre to the other end of the enzyme. (b) The template
chain of DNA. (c) The replica.
Stages of replication: I, “landing” of the enzyme upon the edge of template DNA;
II, the daughter chain appears shorter than the template at its Y-end by 1, because of
marginotomy; III, the enzyme leave-s the daughter chain which is not completed by the
length I,, at its 3’end; IV, the enzyme is dissociated from the template. Latin numerals
in all f&urea (Figs 1 to 4) denote successive stages of DNA replication. Horizontal arrows
showthe direction of migration of the enzyme from 3’-end of the template.
nucleotide chain. (The physical basis for such a mechanism could be that
supporting and motive subunits belonging to the segment responsible for
its movement, are situated at the opposite ends of the segment.) That is
why the catalytic centre of a mono-block DNA-polymerase cannot copy a
template exactly from the first to the last of its nucleotides. This results in
formation of replicas which are shortened from 3’- and (or) S-ends. This
type of synthesis, involving shortening of the replica due to catalytic inactivity
of edges in the enzyme molecule (Fig. I), is called polymerase-dependent
marginotomy.
(3) There may be three types of polymerase-dependent marginotomy :
proximal, distal and medial. Marginotomic shortening of replicas at their
5’-end, nearest to the point where the synthesis starts, is called proximal
marginotomy or 5’-marginotomy (Fig. 2); while that occurring at the 3’-end
is distal marginotomy or 3’-marginotomy (Fig. 3). Thus, there are both
types of marginotomy in the example shown in Fig. 1, because polymerase
of type a has two “dead zones”.
 184 A. M. OLOVNIKOV

ho. 2. The scheme of proximal polymemse& pendent marginotomy of DNA. al, The
molecule of a sin&segment DNA-polymerase with a “right-edge” position of the cata-
lytic centre.

FIG. 3. The scheme of distal polymemwdependent marginotomy of DNA. aa, The

molecule of a single-segment DNA-polymerase with a “left-edge” position of the catalytic
cadre.

Medial marginotomy occurs during replication of a broken template and

is expressed as the sum of the two other types of marginotomy. Copying
of template ends located medially, which are formed in the case of breaking
of the replicon, may be accompanied by the usual marginotomic shortening
of the copy of the 3’ and 5’ ends of the template in the region of the break.
However, when the broken ends of the template are fused, the consequences
of such inside-replicon medial marginotomy may be eliminated by a repair
enzyme. (In the absence of a repair enzyme, all medially marginotomized
replicas would appear broken after replication is completed.)
(4) Arrest of migration along the template, when a segment is hanging
over its edge, is typical not only for mono-block DNA-polymerases, but
also for the DNA-repair enzyme molecules, functioning as a single block;
this excludes the possibility of repair at the ends of the double helix. The
same is true of similar enzymes transcribing RNA from RNA or DNA, as
well as of DNA from RNA.
 MARGINOTOMY THEORY OF AGEING 185

(5) Another variant is also possible. This would be an RNA-primer-

dependent 5’-terminal marginotomy, which would occur if DNA-polymerase
of type a, (as in Fig. 3) needs, for initiation of replication, the presence of
an initiating RNA-primer on the 3’-end of the DNA template. The replica
will be shortened at its 5’-end by the length of RNA-primer, which, as RNA,
must be removed from the final product. The 3’-end of the same replica
also will be shortened, by polymerase-dependent marginotomy. The RNA-
primer itself, if it does not come from outside but is synthesized by the
RNA-polymerase from the 3’-end of the template, may be shortened on its
5’-end due to polymerase-dependent marginotomy (providing that RNA-
polymerase is analogous to type a of DNA-polymerase).
(6) Marginotomy will not occur if the synthesis of DNA, occurring
without RNA-primer, is catalysed by tandem-polymerase. In this enzyme,
each segment functions independently of the activity of the other. Two
segments paired on a dimer, each possess a catalytic centre, and both centres
are oriented towards the same template chain. Since each of the segments
functions individually, if one of them protrudes beyond the end of the
polynucleotide chain, movement continues along the template with the
help of the second, still not over-hanging, segment. Hence the catalytic
activity of the “paralysed” segments may be expressed as a result of a
compulsory movement of its catalytic centre along the template. In the
outcome, the replica formed is not marginotomized and corresponds exactly
to the template in length (Fig. 4).
Tandem-DNA-polymerases of different organisms may have different
configurations, as in Fig. 4 where a2 -a, acts without protrusion of any
part of the tandem or it may have other arrangements of segments (see
Figs 1, 2, 3), types a, -a,, a2-a2 and a-a. As shown in Fig. 4, a poly-
nucleotidligase acts in the final stage. Thus, tandem-polymerases are factors
of antimarginotomy. Tandem-polymerase of any of the types mentioned

FIG. 4. The scheme of DNA replication by the tandem-DNA-polymerase not involving

marginotomy. A tandem of a2 - u1 type is shown. The vertical arrow indicates the point
of action of a polynucleotidligase.
T.B. 11
186 A. M. OLOVNIKOV

above have two identical catalytic centres per molecule. Thus they are
“bis-catalytical” tandems.
(7) Another type of tandem, “a bis-translocational” tandem, is also
possible. In this case, the catalytic centre is localized between two indepen-
dently acting translocational segments each of which is capable of producing
the movement of the entire tandem, even if the second translocational
segment protrudes beyond the template’s end and is “paralyzed”. The centre
for recognition of the initiating sites of the template is located only in the
front segment of such a bis-translocational tandem. In this case, the single
catalytic centre can copy both ends of the template but only if the growing
double helix is not an obstacle to the expression of translocational activity
of the rear segment during replication of the 5’ terminus of the template.
(8) Mono-block DNA-polymerase functions in the majority of somatic
cells, namely those which possess limited cell doubling potential. Each
linear chromosome in a normal cell of an eukaryote containing a mono-block
DNA-polymerase is subject to marginotomy, unless the chromosome
possesses circular replicons at its ends. Marginotomy leads to edge deletions
in the end-genes of chromosome. As a means of temporary protection of
vital genetic material in the telomere against the destructive effect of mar-
ginotomy, each chromosome possesses two specialized genes, the so-called
end-genes or telogenes (Muller, 1940), located at the opposite ends of the
DNA mononeme. It is assumed that telogenes bear no genetic information,
but fulfill “buffer” function. In the course of each mitosis, the telogene is
marginotomically shortened by the RNA-primer length and/or by an
appropriate length I,,, (see Figs 1, 2, 3) thus protecting informative genes
from marginotomy. A telogene also has the function of a starting point of
the end-replicon of a chromosome. A telogene consists of a series of starting
sites (or a series of “recognons”); the polymerase starts each time from the
3’-terminal starting site, i.e. from the 3’-recognon. This recognon will not
be reproduced from the 5’- or/and 3’-end of a DNA strand in the daughter
double helix because of marginotomy. After disappearance of the last
recognon of a telogene, the whole end-replicon of telomers in a daughter
mononeme of DNA will not be replicated.
(9) Genes within the end-replicons in chromosomes which neighbour the
last recognon of a telogene, are vital for maintaining various structures and
functions of a cell. Most important among these are genes specifying RNA-
polymerase or other parts of the transcriptional machinery. Disappearance
of end-replicons in chromosomes and, hence, disappearance of the above-
mentioned genes should lead to pathological changes in different cellular
structures and functions, including changes in karyotype, to cell ageing, and
later to death of the cells.
 MARGINOTOMY THEORY OF AGEING 187
(10) Marginotomic death of some dividing cells of eukaryotes, including
clones participating in regulation of the hormonal status of the body (death
of cells participating in regulation of the activity of hypothalamus and of
others homeostatic centres), is the primary cause of ageing in multicellular
organism.
(11) Tandem-DNA-polymerases are present in cells capable of unlimited
division, for example, in tumor cells, in permanent cell lines, in stem cells,
in germ cells, and in some other cases.

3. Experiments Bearing on the Model and Discussion of the

General Mechanism
The proposed theory of marginotomy provides an explanation of the
limited potential for division exhibited by normal somatic cells of eukaryotes,
as by human fibroblasts in monolayer tissue culture (Hayflick, 1965; Green
& Todaro, 1967), which die after 50 + 10 mitoses. Similar results were
obtained in vitro and in uivo with cells and tissues of various origin (Hayflick,
1972; Holeckova & Cristofalo, 1970; Daniel & Young, 1971). The finite
lifetimes of chick embryo fibroblast cultures average 23.5 population
doublings. Normal mouse embryo fibroblasts are reported to be capable of
between 14 and 28 doublings, and 30 doublings are obtained with marsupial
cells. The normal cells from various kinds of rodent tissue serially trans-
planted in vivo also have a limited proliferative capacity.
If during each mitosis the replica shortens by 10-20 nucleotides, then
after 50 doublings it will be shortened by 500-1000 nucleotides or by the
length of an average gene. Following complete exhaustion of the buffer
telogenes sacrificed by the cells, these fibroblasts die.
From Fig. 1 one can determine the future life-span of any clone of cells,
which depends, other things being equal, on the length of telogenes and the
rate of their shortening. The life-span is expressed by the formula
1T
t=K-
( >
--n,
1,
(1)
where r is the life-span of the clone, K is a correlation factor between the
duration of a clone’s life and number of mitoses, IT is the length of the
telogene, i.e. the physical length of the polynucleotide chain of the buffer
end-gene, I,,, is the length of marginotomy and n is the number of mitoses
which have already occurred.
Length Z, of equation (1) should be species-specific, although there may
be individual variations between telogenes, due to deletions and insertions
188 A. M. OLOVNIKOV

in different individuals within the species, which should be reflected in the

duration of their life. An increased length 1, would provide longer life for
clones and organisms. Reduction of Zr is equivalent to decreasing the
life-span. But a limited life-span, other conditions remaining equal, speeds
up evolution (Weismann, 1882). Therefore, telogene length I, should be
one of the major objects of natural selection during the entire course of
evolution, since variations in this length would inevitably influence the
number of simultaneously living generations in a population and, hence,
affect gene circulation and competition between species and within them.
The product of the length of marginotomy and the number of past
mitoses, I,,,n, is an index of “biological age” of the clone. Some difference
in length among telogenes of different chromosomes of the same cell must
lead to asynchronous exhaustion of those telogenes and, hence, to the
successive disappearance of corresponding end-replicons. This circumstance
must be the primary cause of the fundamental fact that clone ageing pro-
gresses gradually and that it is not a one-hit process.
An informative oligonucleotide, built into DNA after a telogene and
controlling synthesis of a repressor of differentiation, might serve as a means
of counting mitoses performed in the course of morphogenesis. Marginotomic
elimination of such an oligonucleotide would present an appropriate signal
for the beginning of further differentiation. Lengthening of the telogene
would increase the number of possible mitoses in differentiation.
It can be suggested that simultaneous occurrence of two processes, (a) a
switching on of an antimarginotomic mechanism, providing the clone with
unlimited cell doubling potential and (b) a switching off, coincidently with
the first process in time, of the mechanism of contact inhibition, comprise
the two necessary and sticient conditions for malignant transformation of
any cell in any organism.
The necessity of preventing the marginotomic reduction of the genome is
the main reason why the chromosome of limitlessly dividing bacteria, that
of many viruses, of mitochondria and of some other objects possess circular
form, or acquire such a form before replication (Cairns, 1963; Fraenkel-
Conrat, 1969; Lark, 1969; Nass, 1969). A ring, which has no ends, is an
antimarginotomic factor, like the tandem-polymerase. When a circular
chromosome is present, even the mono-block, DNA-polymerase does not
shorten replicas.
Some other types of antimarginotomy, besides ring and tandem, are
conceivable, for example (a), (b) and (c) as follows.
(a) Ends of a pair of telogenes may get fused together in a loop between
two neighbouring DNA helixes. This is, possibly, employed by cells of some
vegetatively propagating objects, but is less likely for cells of higher plants
 MARGINOTOMY THEORY OF AGEING 189
and animals because of the ditliculty of separating of such linear-loop
structures. (This type, in fact, is equivalent to a ring.)
(b) A shortened telogene may be built to completion at the expense of
single-stranded DNA fragments coming from outside, which are comple-
mentary to the uncopied part of the template and therefore hybridized
with it.
(c) There may be incorporation of a series of non-complementary nucleo-
tide units into ends of replicas by enzymes of non-template synthesis (stan-
dard length and nucleotide composition of replicas in this case are excluded).
If enzymes of non-template synthesis lengthen the 3’-end of the template
and if DNA-polymerase of type a1 (Fig. 2) begins the synthesis from this
additional site, then the length of the replica will not be shortened relative
to the template’s original length.
However, tandem and ring seem to be main means of antimarginotomy.
Besides others, some immunological problems arise from the concept of
marginotomy and antimarginotomy. The cellular and humoral immune
mechanisms may play some part in ageing of the body not only by direct
damage of target cells, as is usually assumed (Walford, 1969), but also,
obviously, by stimulation of cells to enter mitoses, so they will be eliminated
because of marginotomy. Development of immunological tolerance to an
antigen which can be the specific mitogene would prevent elimination by
marginotomy of a great number of lymphoid cells. Thus immunological
tolerance has, besides its other functions, also the function of immunological
antimarginotomy.
The reduction of immunological potency with the age depends, among
other reasons, also on the age involution of the thymus. However, the
primary cause of involution of the thymus itself has not been explained up
to now (Burnet, 1970). The marginotomy which can cause the elimination
by mitoses of large numbers of cells of different organs may prove to be
responsible for the involution of thymus also.
Antimarginotomy (for instance through fusion of polynucleotides into
rings) should have existed from the very first stage of evolution of life on
the Earth, since otherwise preservation of genetic information in the enzymic-
ally doubling genome would not have been possible.
The principle underlying marginotomy seems to be applicable not only
to enzymes of template synthesis. For example, in hydrolases the distance
from the edge of the enzyme molecule (or from the substrate transporting
device) to the catalytic centre of the enzyme determines, probably, the size
of non-hydrolysable products, that is, under comparable conditions, the
extent of hydrolysis: the smaller this distance in the enzyme, the smaller
will be the size of undigested fragments.
190 A. M. OLOVNIKOV

4. Prediction
It seems quite expedient to search for cellular factors controlling the
mechanisms of marginotomy and antimarginotomy in template synthesis of
polynucleotides, which might repress or derepress, correspondingly, genes
determining monosegment DNA-polymerase and tandem-DNA-polymerase
or other means of antimarginotomy. Such factors (“marginotomites” and
“antimarginotomites”) would, probably, regulate the duration of life of
different cell clones and of the organisms which are composed of them.
It seems also quite important to identify the genes of end-replicons of
chromosomes. Possibly among them will be found the genes involved in
transcriptional machinery. Transduction into chromosomes of additional
doses of such genes as well as artificial lengthening of the telogenes could be
a means of delaying ageing of proliferating cell clones.
Thus, the terminus is the heel of Achilles of the DNA double helix, but
it can be protected by antimarginotomy.

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