Mechanisms of Ageing and Development
109 (1999) 141 – 151
www.elsevier.com/locate/mechagedev

Age-dependent changes in DNA polymerase
fidelity and proofreading activity during cellular
aging
Mitsugu Fukuda a, Takahiko Taguchi a,*, Mochihiko Ohashi b
a

Department of Gene Regulation and Protein Function, Tokyo Metropolitan Institute of Gerontology,
35 -2 Sakae-cho, Itabashi-ku, Tokyo 173 -0015, Japan
b
Cancer Institute Hospital, Japanese Foundation for Cancer Research, 1 -37 -1 Kami-Ikebukuro,
Toshima-ku, Tokyo 170 -0012, Japan
Received 26 January 1999; received in revised form 20 May 1999; accepted 21 May 1999

Abstract
DNA polymerase h and the 3%“ 5% exonuclease involved in the proofreading of DNA
synthesis were isolated from human diploid fetal lung fibroblast (TIG-1) cells at various
population doubling levels (PDL). The final PDL of the TIG-1 cells used in these experiments was 70. The fidelity of DNA polymerase h remained high until late passage and fell
suddenly just before the end of the life span between 65 and 69 PDL. The activities of the
3%“5% exonuclease related to proofreading remained unchanged from 21 to 61 PDL, but the
activity decreased rapidly in more aged cells. The 3%“ 5% exonuclease activity at 69 PDL was
about 50% of that in TIG cells at 21 PDL. In vitro DNA synthesis by DNA polymerase h
from TIG-1 cells harvested at 69 PDL showed the amount of non-complementary nucleotides incorporated to be decreased by the addition of the 3%“ 5% exonuclease from the same
cells. However, not all errors were edited out since the ratio of DNA polymerase activity to
3%“5% exonuclease activity was adjusted to reflect that in vivo and the infidelity of DNA
synthesis by error-prone DNA polymerase h from aged cells was improved by the addition
of the highly active 3% “5% exonuclease from cells at 41 PDL. These results suggested that the
mutation frequency rises just before the end of the life span of TIG-1 cells. © 1999 Elsevier
Science Ireland Ltd. All rights reserved.
Keywords: DNA polymerase; 3%“ 5% exonuclease; Fidelity; Proofreading; TIG-1 cell; Replicative cellular
aging

* Corresponding author. Tel.: +81-3-39643241; fax: +81-3-35794776.
0047-6374/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 3 4 - 2

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1. Introduction
It is well known that non-malignant cells have a finite life span in culture. Many
researchers have utilized early and late passage cultured cells as a model system for
studying cellular aging in vitro. Cellular aging and organismal aging may differ.
Cellular aging has been mainly studied with respect to the replicative life span using
fibroblasts, whereas, many cells that comprise tissues and organs in animals are
post-mitotic cells and age-associated changes have been observed in these tissues
and organs. However, it is very important to clarify the mechanism of finite life
span and to understand the characteristics of permanent cell lines.
The expressions of 11 cell cycle-dependent genes in senescent WI-38 fibroblasts
were studied and compared with the results obtained in early passaged WI-38 cells
(Rittling et al., 1986). The results indicated that senescent, noncycling cells are
blocked at the G1/S boundary. After that, a gene coded senescent cell-derived
inhibitor (Sdi1) that exhibits DNA synthesis-inhibitory activity was found (Noda et
al., 1994). The expression of sdi1 is 10- to 20-fold higher in senescent cells than in
young cells. The Sdi1 protein is called the p21 protein and DNA synthesis is
inhibited by the coupling of p21 and PCNA (Waga et al., 1994). Moreover,
telomeres have been identified as oligonucleotide repeats related to a limited
potential for cell proliferation. A gradual loss of telomeric DNA occurs during
aging as has been reported (Harley et al., 1990; Hastie et al., 1990; Chang and
Harley, 1995), but immortal cells show no net loss of telomere length (Counter et
al., 1992). On the other hand, in the synthesis of telomeric DNA, it is thought that
first telomeric DNA is polymerized by a specific telomere terminal transferase
(Greider and Blackburn, 1985). Greider and Blackburn (1987) have found that the
telomere terminal transferase is a ribonucleoprotein enzyme, and then, that telomerase is a kind of reverse transcriptase has been reported by Blackburn (1991).
Telomerase activity was detected in immortal cancer cells but not in normal somatic
cells (Kim et al., 1994). These data suggest that the length of telomeres is related to
cellular mortality. These results indicate that the expression of the Sdi1 protein and
loss of telomeric DNA can be used to explain replicative cellular aging.
On the other hand, the accurate transfer of genetic information to progeny cells
is very important in maintaining cellular specificity. Therefore, DNA must be
duplicated in each generation with high fidelity. However, the fidelities of DNA
polymerases from late passage cultured cells (Fry and Weisman-Shomer, 1976; Linn
et al., 1976; Murray and Holliday, 1981; Krauss and Linn, 1982) and from tissues
of aged animals (Srivastava et al., 1991, 1993; Taguchi and Ohashi, 1996, 1997) are
decreased. Prokaryotic DNA polymerases and eukaryotic DNA polymerases k, l,
and m possess a 3% “ 5% exonuclease activity for proofreading (Wang, 1991). Therefore, the fidelities of DNA synthesis by DNA polymerase k, l, and m are involved
in proofreading (Kunkel et al., 1987; Thomas et al., 1991). In contrast, most
purified DNA polymerases h and i lack the 3%“ 5% exonuclease activity (Wang,
1991). However, to clarify the effect of proofreading on the fidelity of DNA
synthesis by DNA polymerase h or i is very important in understanding aging and
mutation. We have already reported the presence of a 3% “5% exonuclease related to

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143

the proofreading of DNA synthesis by DNA polymerases h and i (Taguchi et al.,
1998b). This 3% “5% exonuclease activity decreases in rat liver during aging (Taguchi
et al., 1998c). It is possible that both the increased misincorporation by DNA
polymerases and the decline in proofreading activity lead to an increase in the rate
of mutations on DNA in aged rats.
In this paper, we compared the fidelity levels of DNA synthesis by the coexistence of DNA polymerase h and the 3% “ 5% exonuclease prepared from early
passaged TIG-1(Ohashi et al., 1980) and from aged cells harvested just before the
end of their life span. Further, the appearance of mutations and cellular aging will
be discussed.

2. Materials and methods

2.1. Cells and cell culture
TIG-1 cells were cultured in plastic dishes (Falcon, 55 cm2) containing 10 ml of
basal medium (Eagle, GIBCO) supplemented with 10% fetal bovine serum
(GIBCO) at 37°C under humidified 5% CO2 –95% air. The cells were subcultured at
a 1:4 split ratio once a week as described previously (Ohashi et al., 1980).

2.2. Chemicals and compounds
Chemicals were purchased as follows: deoxynucleoside triphosphates from
Boehringer Mannheim-Yamanouchi (Tokyo, Japan); [3H]deoxynucleoside triphosphates from Dupont/New England Nuclear (Boston, MA); poly dA-dT10 and poly
dA-d(T9-C) from P-L Biochemicals (Milwaukee, WI).

2.3. Isolation of DNA polymerases h and the 3 %“ 5 % exonuclease in6ol6ed in
proofreading
TIG-1 cells at various population doubling levels (PDL) were harvested and cell
numbers were determined with a hemocytometer. The harvested cells were washed
with 0.85% NaCl and suspended in about nine volumes of 0.005 M Tris-HCl buffer,
pH 8.0, containing 0.34 M sucrose, 25 mM KCl, and 5 mM MgCl2 (buffer A).
TIG-1 cells suspended in buffer A were sonicated for 1 min with a Sonifier Cell
Disrupter (Model 185, Branson Sonic Power Co., New York, NY). The sonicates
were centrifuged at 100 000× g for 120 min and the supernatants were used as
crude DNA polymerase extracts. The partial purification of DNA polymerases
from the extracts derived from TIG-1 cells was carried out by the following
procedure. One milliliter of the crude enzyme extract was dialyzed against 0.05 M
phosphate buffer, pH 6.5, containing 1 mM 2-mercaptoethanol (buffer B) and
applied to a phosphocellulose column (0.5× 2 cm) equilibrated with buffer B. The
column was washed with 3 ml of buffer B and developed with a 30 ml linear
gradient of 50 – 400 mM phosphate buffer containing 1 mM 2-mercaptoethanol.

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One milliliter fractions were collected. An aliquot from each fraction was assayed
for DNA polymerase activity and 3% “ 5% exonuclease activity. The pass through
fraction was dialyzed against 0.01 M Tris-HCl buffer, pH 8.0, containing 50 mM
KCl and 1 mM 2-mercaptoethanol (buffer C) and applied to a DEAE-cellulose
column equilibrated with buffer C. The column was washed with 3 ml of buffer C
and developed with a 30 ml linear gradient of 50 mM to 1.0 M KCl in the same
buffer. One milliliter fractions were collected.

2.4. Standard assay method for DNA polymerase acti6ity
DNA polymerase activity was measured by the method of Taguchi and Ohashi
(1996).

2.5. Assay of the fidelity le6els of DNA polymerases
The incorporation of complementary nucleotides into a template-primer was
measured. The reaction mixture contained 12.5 nmol unlabeled deoxythymidine
5%-triphosphate (dTTP), 12.5 mCi/1.01 nmol of [3H]dTTP, 1.0 mmol of MgCl2, 1.5
mmol of dithiothreitol, 2.5 mmol of Tris-HCl buffer, pH 8.3, 20 mg of poly dA-dT10,
and 25 ml of enzyme fraction in a final volume of 125 ml. The reaction was initiated
by adding the enzyme fraction and the mixture was incubated at 37°C for 60 min.
The amount of non-complementary nucleotide incorporated into poly dA-dT10
was measured in the same reaction mixture except that 12.5 mCi of non-complementary [3H]deoxycytidine 5%-triphosphate (dCTP) or [3H]deoxyguanocine 5%-triphosphate (dGTP) was used instead of the complementary radioactive nucleotide.
All other assay procedures and the calculation of fidelity were performed as
described previously (Taguchi and Ohashi, 1996).

2.6. Assay of exonuclease acti6ity and proofreading acti6ity
Assays of exonuclease activity and proofreading activity were measured by the
method of Taguchi et al. (1998b).
3. Results
The TIG-1 cells were harvested at a semi-confluent state and DNA polymerase
and 3% “5% exonuclease were isolated from the TIG-1 cell extracts by phosphocellulose column chromatography. DNA polymerase was eluted by buffer B containing
about 0.18 M KCl (Fig. 1). From our previous data, the DNA polymerase eluted
at this KCl concentration was estimated to be h type. In fact, this DNA polymerase
preparation was inhibited by aphidicolin, and sedimented at an S value of 7.5 in a
5–20% linear sucrose density gradient (w/w) containing 0.3 M KCl (data not
shown). Furthermore, the DNA polymerase activity and none of the 3% “ 5%
exonuclease activity corresponded (Fig. 1). These characteristics are those of DNA
polymerase h itself.

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Two large peaks and a minor peak possessing 3% “5% exonuclease activity were
observed (Fig. 1). The 3% “5% exonuclease related to proofreading from rat liver
(Taguchi et al., 1998b) eluted in the first peak. The proofreading activity was not
detected in the other 3% “5% exonuclease fractions (data not shown). Therefore,
fractions 2 – 8 from phosphocellulose column chromatography were mixed and
rechromatographed on a DEAE-cellulose column. Three peaks of 3%“ 5% exonuclease activity were observed. The 3% “ 5% exonucleases eluted in the pass through
fraction, at 0.35 M KCl, and at 0.55 M KCl in buffer C (Fig. 2). These fractions
are called 3% “ 5% exonucleases according to their order of elution, DE-1, DE-2, and
DE-3, respectively. DE-3 was not found in the liver of control rats (Taguchi et al.,
1998b). Thus, the 3% “ 5% exonucleases can be separated into five molecular species
by phosphocellulose and DEAE-cellulose column chromatographies. DNA synthesis in the presence of DNA polymerase h and each of the 3% “ 5% exonuclease
activities obtained by DEAE-cellulose column chromatography on poly dA-d(T9-C)
as a template-primer was measured. The amount of DNA synthesis by the
coexistence of DNA polymerase h and DE-2 on poly dA-d(T9-C) was similar to the
amount synthesized in the presence of only DNA polymerase h on dA-dT10 (Table
1). This indicates that DE-2 is involved in proofreading.
The fidelities of DNA polymerases extracted from in vitro cultured cells decrease
with increasing PDL (Fry and Weisman-Shomer, 1976; Linn et al., 1976; Murray
and Holliday, 1981; Krauss and Linn, 1982; Taguchi et al., 1998a). These do not
include any added factor for proofreading. Therefore, it is important to clarify the
effect of the proofreading activity on the replicative life span of cultured human
fibroblasts. First, the change in DE-2 activity during in vitro cellular aging was
studied. The activity of DE-2 decreased gradually in TIG-1 cells from 25 to 61

Fig. 1. Separation of DNA polymerase and 3%“ 5% exonucleases by phosphocellulose column chromatography of extracts of TIG-1 cells at 21 PDL. Chromatography was carried out as described in Section 2.
The concentration of phosphate in elution buffer B is indicated by the line (— ). DNA polymerase
activities ( — ) are expressed as pmol of [3H]dTMP incorporated into activated DNA. 3%“ 5%
exonuclease activities ( — ) are expressed as pmol of hydrolyzed [3H]DNA.

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Fig. 2. Separation of 3%“ 5% exonuclease activities by DEAE-cellulose column chromatography. Aliquots
(7 ml) of fractions 2–8 from phosphocellulose column chromatography (Fig. 1) were equilibrated with
buffer C and applied to a DEAE-cellulose column. The chromatographic conditions and the assay for
3% “5% exonuclease activity ( — ) in each fraction were as described in Section 2. The concentration
of KCl in elution buffer C is indicated by the line (—).

PDL, and then decreased rapidly with increasing PDL thereafter (Fig. 3). The final
population doublings of TIG-1 cells used in these experiments was 70. On the other
hand, the fidelities of DNA polymerase h from early passaged TIG-1 cells (21 PDL)
were high (Table 2) and the fidelity in middle passage cells maintained this high
level (data not shown). However, the fidelities of DNA polymerase h from TIG-1
cells just before the end of the life span (69 PDL) was very low (1/5 700–1/7 200)
as shown in Table 2. The effect of the 3%“ 5% exonuclease, DE-2, on the fidelity of

Table 1
Proofreading activities of 3%“5% exonucleases DE-1, DE-2, and DE-3a
DnNA polymerase

Nuclease

[3H]dTMP incorporated (pmol)
Template-primer
Poly dA-dT10

h
h
h
h

–
DE-1
DE-2
DE-3

Poly dA-d(T9-C)

3.85
3.77
4.05
4.09

0.15
0.22
3.44
0.14

a
DNA polymerase h was collected from fractions 16–21 of the phosphocellulose column chromatography shown in Fig. 2. DE-1, DE-2, and DE-3 were collected from fractions 2–8, fractions 16–19, and
fractions 24–26, respectively, of the DEAE-cellulose column chromatography shown in Fig. 3. The assay
conditions were the same as described previously (Taguchi et al., 1998a).

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147

Fig. 3. Changes in the 3% “ 5% exonuclease activities of TIG-1 cells at various PDL. DE-2 was separated
from TIG-1 cells at 21, 29, 41, 49, 53, 57, 61, and 69 PDL, and the activities were plotted. The plotted
DE-2 activities ( — ) are expressed as pmol of hydrolyzed [3H]DNA per 107 cells.

DNA synthesis by error-prone DNA polymerase h from 69 PDL cells was
examined. When DE-2 from 69 PDL cells was added to the reaction mixture for in
vitro DNA synthesis, the fidelity of DNA synthesis increased. A greater increase in
fidelity was observed by the addition of the highly active 3% “ 5% exonuclease from
41 PDL cells (Table 2). However, recovery from misincorpolation was not
complete.

Table 2
Effect of 3% “5% exonuclease DE-2 on the fidelity of DNA synthesis by DNA polymerase h a
Enzymesb
Pol h

Template-primer: poly dA-dT10
DE-2

Non-complementary nucleotide incorporated
dCMP

+(21)
+(41)
+(69)
+(69)
+(69)

–
–
–
+(69)
+(41)

dGMP

B1/87 000
B1/85 000
1/7 200
1/11 000
1/29 100

B1/87 000
B1/85 000
1/5 700
1/12 500
1/43 600

DNA polymerase a and DE-2 were prepared by the methods described in Section 2. The calculation
of fidelity and the assay conditions were the same as described previously (Taguchi et al., 1998a) except
that 70 ml of DNA polymerase fraction and 30 ml of DE-2 were added to the reaction mixture instead
of 50 ml of each enzyme.
b
Numbers in parentheses are the PDL of the TIG-1 cells used as the enzyme source.
a

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4. Discussion
The 3% “5% exonucleases were separated into five species by phosphocellulose
and DEAE-cellulose column chromatographies. The minor peak eluted at about
0.2 M KCl in buffer B from the phosphocellulose column and the third peak
eluted at 0.55 M KCl in buffer C from the DEAE-cellulose column have not
been found in extracts of rat liver and regenerating rat liver. The proofreading
activity was assayed by measuring the DNA synthesizing activity after removing
mispaired nucleotides at the 3%-primer terminus in the presence of DNA polymerase and the 3% “5% exonuclease. The 3% “ 5% exonuclease related to proofreading in TIG-1 cells of human origin elutes at about 0.39 M KCl in buffer C by
DEAE-cellulose column chromatography. We have reported that the 3%“ 5% exonuclease involved in proofreading from rat liver also elutes in the 0.4 M KCl
fraction by the same chromatography. Therefore, both activities are named DE2. It is possible that the DE-2 enzymes of human and rat origin are the same
kinds of enzyme based on their similar properties, elution profile from the two
ion-exchange column chromatographies mentioned above, structures of the reaction products, requirements for divalent cations, pH optima, and inhibition
profiles by ATP (data not shown). As both enzymes have similar characteristics,
we conclude that the human DE-2 and the rat DE-2 are the same enzyme.
Since it has been reported that human diploid fibroblasts have a finite life
span in culture, they have been widely used as a model system for aging research
in vitro. The replicative life span is explained by the expression of Sdi protein
(Noda et al., 1994) and the gradual loss of telomere length (Harley et al. 1990;
Hastie et al., 1990). On the other hand, changes in the fidelity of DNA polymerases during aging have been studied as one field of aging research. Decreased
fidelities of DNA polymerase during in vitro aging (Linn et al., 1976; Murray
and Holliday, 1981; Krauss and Linn, 1982; Taguchi et al., 1998a) and in chick
embryo cells (Fry and Weisman-Shomer, 1976) have been reported. Furthermore,
the fidelities of DNA polymerases from aged mice and rats also decrease (Srivastava et al., 1991; Srivastava and Busbee, 1992; Srivastava et al., 1992, 1993;
Taguchi and Ohashi, 1996, 1997; Taguchi et al., 1998a). However, prokaryotes
and eukaryotes have a function that prevents misincorporations during DNA
synthesis. Most prokaryotic DNA polymerases and DNA polymerases k, l, and
m possess a 3% “ 5% exonuclease activity within the molecule. Although DNA
polymerases h and i do not contain a 3% “ 5% exonuclease activity, DNA synthesis proceeds accurately with the coexistence of error-prone DNA polymerase h or
i and a separate 3% “5% exonuclease related to proofreading in vivo (Taguchi et
al., 1998b,c). These experiments were performed using rat liver, and it has been
suggested that the presence of a proofreading system is very important for
maintaining genetic information in vivo. A decline in proofreading activity
should result in an increase in the mutations in progeny cells. This may be lead
to the suspension of cellular proliferation and to cell death. Therefore, the activity of the proofreading system should be studied during in vitro aging. In these

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experiments, the fidelity of the DNA polymerase h from TIG-1 cells at 69 PDL
was decreased as previously reported (Linn et al., 1976; Murray and Holliday,
1981; Krauss and Linn, 1982; Taguchi et al., 1998a). Although the DE-2 activity
from early and middle passaged TIG-1 cells remains high, that from TIG-1 cells
just before the final population doublings decreases rapidly (Fig. 2). The fidelity
of DNA synthesis by error-prone DNA polymerase h from cells at 69 PDL can
be partially recovered by adding DE-2 from the same cells (Table 2). This
indicates that the mismatch mutations probably occur in the daughter cell DNA
during replication of the 69 PDL cells. However, fidelity was improved by the
addition of more active DE-2 from middle passaged cells (41 PDL). This suggests that the decrease in DE-2 activity as well as a decrease in DNA polymerase h fidelity lead to mismatch mutations.
Mismatch mutations may be removed by DNA repair. However, unscheduled
DNA synthesis at late passages of UV-irradiated WI-38 cells decreased (Hart
and Setlow, 1976). In addition to decrease in DNA polymerase h fidelity and
proofreading activity as mentioned above, the decrease in DNA repair activity in
the cells before the final population doubling should rise mutation frequency.
We must discuss the cause for the appearance of error-prone DNA polymerases and the loss of DE-2 activity. We have no evidence related to the
occurrence of such altered enzymes. One possibility is that the mutation in the
genes for these enzymes may be caused by DNA damage such as oxidation.
TIG-1 cells are serially cultured under the usual conditions and have not received any accident such as radiation, mutagen, or carcinogen treatment. Under
such conditions, oxidation of the enzyme proteins or genomes is most probable.
In fact, oxidative damage increases in human diploid fibroblasts during in vitro
aging (Homma et al., 1994; Chen et al., 1995; Hirano et al., 1995) and in vivo
aging (Fraga et al., 1990; Kaneko et al., 1996). Furthermore, oxygen radical
induced mutagenesis of DNA polymerase in vitro has been reported (Feig and
Loeb, 1994).
In late passaged cell, the decreases in the fidelity of DNA polymerase h and
proofreading activity must lead to mutations in the DNA. Misincorporations by
DNA polymerase are basically random although some hot spots may be present.
If misincorporations occur in the gene encoding part of the machinery involved
in cellular DNA replication, it is possible that the cells will be unable to proliferate due to replicative trouble. Furthermore, if a mutation occurs in a gene
essential for survival, the cell may die. Such accidents may be related to the
finite life span of cultured cells.

Acknowledgements
The authors would like to express their gratitude to Dr. Margaret DooleyOhto for assistance with the manuscript. This work was supported in part by a
project grant (for research on Parameters of Biomedical Aging) from the Institute of Physical and Chemical Research, Japan.

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