Journal of Gerontology: BIOLOGICAL SCIENCES
2003, Vol. 58A, No. 3, 205–211
Copyright 2003 by The Gerontological Society of America
Age-related Base Excision Repair Activity in
Mouse Brain and Liver Nuclear Extracts
Gabriel W. Intano,1 Eun Ju Cho,1 C. Alex McMahan,2 and Christi A. Walter1,3
2
1
Department of Cellular & Structural Biology, and
Department of Pathology, The University of Texas Health Science Center at San Antonio.
3
South Texas Veterans Health Care System, Audie L. Murphy Hospital, San Antonio.
To assess DNA repair activity relative to age, in vitro base excision repair assays were performed
using brain and liver nuclear extracts prepared from mice of various ages. An 85% decline in
repair activity was observed in brain nuclear extracts and a 50% decrease in liver nuclear extracts
prepared from old mice compared with 6-day-old mice. Brain nuclear extracts prepared from old
mice showed a decreased abundance of DNA polymerase-b, but the addition of purified protein
did not restore base excision repair activity. Abundances of other tested base excision repair
proteins did not change relative to age. The conclusion is that, during aging, a decline in DNA
repair could contribute to increased levels of DNA damage and mutagenesis.
W
HILE it is clear that organisms age and develop agerelated pathologies such as cancer, conclusive
identification of the mechanisms involved in aging remain
elusive. More than 40 years ago, Failla (1) and Szilard (2) proposed the Somatic Mutation Theory of Aging. The theory
proposes that a gradual accumulation of mutations occurs
with increasing age, thereby leading to cellular dysfunction
and a breakdown of homeostasis. A role for genetic instability in carcinogenesis, largely an age-related pathology,
is now well accepted (3), but whether genetic instability contributes substantially to overall organismal aging is not clear.
Notably, correlative data support the Somatic Mutation
Theory of Aging (4–10). Later, Alexander (11) developed the
DNA Damage Theory of Aging to explain aging based on
accumulation of DNA damage: a theory that has been supported by correlative data (12–15).
Because DNA repair is tightly linked with levels of DNA
damage and mutagenesis, many studies have focused on the
potential role of DNA repair in aging. Indirect assessment
of DNA repair has dominated studies designed to examine
the potential role of decreased DNA repair in aging. These
studies have included measurements of chromosomal
aberrations (4,16), unscheduled DNA synthesis (17),
abundances of DNA lesions (12–14,18,19), the activity of
specific DNA repair proteins (13,20,21), and the ability to
remove induced damage (19,22). In general these studies
have revealed decreased DNA repair activity with increased
age. Because accumulation of spontaneous DNA damage
and mutations has been hypothesized to contribute to aging,
and because the base excision repair (BER) pathway is the
pathway that largely ameliorates such damage (23), BER
may play an important role in aging. In general, BER
involves the action of specific DNA glycosylases that
catalyze hydrolysis of the N-glycosylic bond of damaged
bases leaving apurinic/apyrimidinic (AP) sites in DNA.
Some DNA glycosylases are bifunctional and nick the
phosphodiester backbone 39 of the abasic site (24). Most
abasic sites are next processed by AP endonuclease (Ape),
which nicks the phosphodiester backbone 59 of the abasic
site (25,26). A DNA polymerase fills in the resulting gap
(27,28). The 39-terminus of the original nick is processed to
leave a 59 phosphate group as a suitable terminus for DNA
ligase to complete repair of the damaged DNA strand by
joining of the 59 phosphate to the 39 hydroxyl group of the
newly replaced bases (27).
Short-patch and long-patch BER pathways have been
described. During short-patch BER, DNA polymerase b (bpol) incorporates a single nucleotide into the strand undergoing repair (28), and DNA ligase III, with its partner Xrcc1,
restores the integrity of the phosphodiester backbone (29).
Long-patch BER utilizes PCNA and DNA polymerase d or
e to incorporate 2–6 nucleotides in the strand undergoing
repair (30). Fen-1 may be involved in removing the displaced flap (31), and DNA ligase I rejoins the phosphodiester backbone (32). It has been suggested that short-patch
BER is more active than long-patch BER in vivo (25).
To test if BER activity changes with age, an in vitro uracil
DNA glycosylase (UDG)-initiated BER assay was used to
examine short-patch BER. Repair activity was examined in
nuclear extracts prepared from brain and liver of neonatal
(6- and 8-day-old [d/o]), young adult (3-month-old [m/o]),
middle-aged (16-m/o), and old (28-m/o) mice.
METHODS
Animals
Six- and 8-d/o neonatal and 3-m/o young adult male
CD1 mice and neonatal B6D2F1 mice were obtained from
Charles River. Young adult (3-m/o), middle-aged (16-m/o),
and old (28-m/o) male B6D2F1 mice were obtained from
the National Institute on Aging. All mice were housed
in a specific-pathogen-free, American Association for the
Accreditation of Laboratory Animal Care-accredited animal
205
206
INTANO ET AL.
facility and fed standard mouse lab chow and water ad
libitum. Adult mice were euthanized using isofluorane
followed by cervical dislocation, while neonatal mice
were overdosed with isofluorane. Tissues were rapidly
removed and used immediately for preparation of nuclear
extracts.
Nuclear Extracts
Brain and liver nuclear extracts were prepared as
described by Widen and Wilson (33), with modification as
described previously (34,35). Luciferase (2.5 ng/ll) was
added to the nuclear preparations just prior to lysis and
subsequently used to assess protein recovery. Immunoglobulin was used as a standard in the Bradford assay (36) to
determine overall protein concentrations according to the
manufacturer’s recommendations (BioRad, Hercules, CA).
Nuclear extracts were separated into single-use aliquots at
10 mg/ml and stored at 2808C until use. Luciferase assays
were performed on nuclear extracts prior to use in repair
assays or Western analyses, as described previously (34,
35), by adding 2 ll of nuclear extract to luciferase buffer
(60 mM Tris-acetate [pH 7.5], 2.5 mM EDTA, 12 mM Mg
acetate, 60 mM dithiothreitol, 5 mM ATP, 0.075% BSA,
and 150 lM luciferin), measuring relative light units (RLUs)
on a Lumat LB 9501 luminometer (Berthold), and comparing the RLUs to a luciferase standard curve (37,38).
UDG-BER Assay
The UDG-BER assay was performed as described
previously (28,34,35). Routinely, 40 lg of somatic tissue
nuclear extract was added to a reaction mix consisting of
3 pmol of a 51-mer oligonucleotide containing a single G:U
mismatch and a 59 fluorescein label on the U-containing
strand (Integrated DNA Technologies) and reaction buffer
(100 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM MgCl2,
1 mM dithiothreitol, 2 mM ATP, 0.5 mM NAD, dATP,
dGTP, and dTTP at 20 lM each, 5 mM ditrisphosphocreatine, 10 units of creatine phosphokinase, 20 nM of unlabeled
dCTP, 20 lCi of [a-33P]dCTP (3000 Ci/mmol)). Samples
were incubated for 10 minutes at 378C. Reactions were
stopped by the addition of stop solution (50 mM EDTA, 0.3
M NaCl, and 80% formamide) and placed on ice. Samples
were then subjected to denaturing polyacrylamide (12%)
gel electrophoresis. Fluorescently labeled oligonucleotide
standards encompassing the linear range of fluorescent
quantification were simultaneously run on each gel. The recovered oligonucleotide was visualized and quantified on a
ChemiImage 4400 (Alpha Innotech, San Leandro, CA) while
radionucleotide incorporation was measured using a GS-363
Molecular Imager System (BioRad).
Enrichment assays were performed by adding 1.25 to
20 ng of Escherichia coli udg (Life Technologies, Grand
Island, NY), human DNA polymerase-b (b-POL), human
APE/REF-1 (Trevigene, Gaithersburg, MD), or murine DNA
ligases I and IIIb (A. Tomkinson, University of Texas
Health Science Center Institute of Biotechnology, San
Antonio, TX) independently to nuclear extracts in 1 ll
volumes. Afterward, UDG-BER assays were performed as
described above.
Western Blot Analysis
Western blot analyses were performed as described (34,
35). Briefly, nuclear extracts prepared from brain and liver
obtained from neonatal, young, middle-aged, and old mice
were separated using SDS-PAGE on a 10% gel (acrylamide:
bis-acrylamide 29:1), followed by electroblotting onto
Trans-Blot Transfer Medium (BioRad). Blots were cut into
three sections based on molecular mass to facilitate
detection of specific antigens. DNA ligases I and III were
detected by rabbit polyclonal antiligase I and ligase III
antibodies (A. Tomkinson, University of Texas Health
Science Center at San Antonio, San Antonio, TX),
respectively. Xrcc1 was detected with rabbit anti-hXRCC1
polyclonal antibody (Serotec, Raleigh, NC). Detection of
Ape/Ref-1 and b-pol were facilitated by the use of rabbit
anti-hAPE/REF-1 (Novus Biologicals, Littleton, CO) and
rabbit polyclonal anti-b-Pol (S. Wilson and R. Sobol,
NIEHS, Research Triangle Park, NC), respectively. Purified
b-POL and APE/REF-1 (Trevigen, Gaithersburg, MD) and
DNA ligases I and III (A. Tomkinson, UTHSCSA, San
Antonio, TX) were included as standards and controls. Goat
antirabbit antibody conjugated to horseradish peroxidase
(Pierce, Rockford, IL) served as secondary antibody.
Visualization was achieved using enhanced chemiluminescence (ECL, Pierce, Rockford, IL). A ChemiImager 4400
(Alpha Innotech) was used to measure intensity of chemiluminescent bands as an integrated density value (IDV).
Statistical Analysis
UDG-BER and western blot data were analyzed using
analysis of variance. Comparisons among means were Bonferroni adjusted. Changes in UDG-BER activity of nuclear
extracts after the addition of purified BER proteins were
compared using Dunnett’s test. P values are presented from
analysis of log-transformed data, whereas means and standard errors computed from untransformed data are presented.
P values ,.05 were considered significant.
RESULTS
In Vitro BER Activity in Brain and
Liver Nuclear Extracts
UDG-BER activity was greater in liver nuclear extracts
than brain nuclear extracts from mice in each of the ages
that were tested: neonatal, young, middle-aged, and old ( p
, .05; Figure 1A and B). UDG-BER activity was slightly
but significantly lower in liver nuclear extracts prepared
from 3-m/o mice compared with 6-d/o mice ( p , .05).
Further reduction in UDG-BER activity was observed in
liver nuclear extracts prepared from 16- and 28-m/o mice,
such that a 50% decrease in UDG-BER activity was detected
between liver nuclear extracts prepared from neonatal and
old mice (Figure 1A).
The only significant difference ( p , .05) in UDG-BER
activity between CD1 and B6D2F1 mice was observed for
brain nuclear extracts prepared from 8-d/o mice: Activity
was approximately 35% lower in CD1 samples than in
B6D2F1 samples. Brain nuclear extracts prepared from
3- and 16-m/o mice displayed 36% of the UDG-BER
SOMATIC BER DECLINES WITH AGE
207
Figure 2. Western blot analysis of base excision repair proteins in nuclear
extracts prepared from brain and liver of 3-month-old (m/o) [3], 16-m/o [16],
and 28-m/o [28] male mice. Bands corresponding to DNA ligase I (130 KDa),
DNA ligase III (93 KDa), Xrcc1 (69 KDa), b-pol (39 KDa), and Ape/Ref-1
(37 KDa) proteins were visualized. A molecular mass protein standard (KDa)
and purified DNA ligases I and III, b-pol, and Ape (STD) are shown for
comparison.
Figure 1. Uracil DNA glycosylase-initiated base excision repair (UDG-BER)
activities. A: Activity detected in nuclear extracts prepared from liver obtained
from 6-day-old [d/o], 8-d/o, 3-month-old [m/o], 16-m/o, and 28-m/o male mice.
Results are presented as means (6 SEM) of three replicate assays for each of
three independent nuclear extract preparations. [a] Significantly less than 6-d/o.
[b] Significantly less UDG-BER activity than 8-d/o. [c] Significantly less than
3-m/o. B: Activity detected in brain nuclear extracts. Results are presented as
means (6 SEM) of three replicate assays for each of three independent nuclear
extract preparations. [a] Significantly less than 6-d/o. [b] Significantly less than
6- and 8-d/o. [c] Significantly less than all earlier stages. BDF 5 B6D2F1 hybrid
mice; CD1 5 CD1 outbred mice.
activity of 6- and 8-d/o mice ( p , .05; Figure 1B). UDGBER activity in brain nuclear extracts obtained from 28-m/o
mice was 14% of that observed for neonatal mice. Overall,
brain nuclear extract UDG-BER activity was observed to
decline approximately 85% between neonatal and old mice.
BER Protein Abundances in Nuclear Extracts
Western blot analysis was used to determine the
proportional abundances of BER proteins in nuclear extracts
prepared from liver and brain isolated from young, middle
aged, and old mice (Figure 2). The proportional abundances
of DNA ligases I and III, Xrcc-1, b-pol, and Ape in nuclear
extracts from liver did not change significantly with increased age (Table 1). Likewise, no significant changes in
the proportional abundances of DNA ligase I and III, Xrcc-1,
and Ape in brain nuclear extracts prepared from different
age mice were detected (Table 2). The abundance of b-pol
in brain nuclear extracts from 28-m/o mice was reduced by
70% ( p , .05) and 40% (not statistically significant)
compared with samples prepared from 3- and 16-m/o mice,
respectively.
Limiting BER Enzyme Activities in Brain and
Liver Nuclear Extracts
To determine if the abundance of a particular protein
limited the UDG-BER activity in brain and liver nuclear
extracts prepared from young and old mice, individual BER
proteins were added to the extracts, which were subsequently assayed for activity. Addition of udg, APE/REF1, b-POL, and DNA ligase III did not significantly alter
UDG-BER activity in liver nuclear extracts prepared from
3- or 28-m/o mice (Figure 3). Likewise, UDG-BER activity
was not affected by the addition of up to 20 ng purified udg,
Table 1. Proportional Abundances of BER Proteins in Nuclear Extracts
Prepared From Liver Obtained From Different Aged Mice
Protein
DNA Ligase I
DNA Ligase III
Xrcc1
b-pol
Ape/Ref1
3-Month-old mice 16-Month-old mice 28-Month-old mice
491.8 6 36.4*
(43.8%)y
57.5 6 11.8
(5.1%)
15.4 6 1.2
(1.4%)
299.1 6 42.0
(26.6%)
259.3 6 38.0
(23.1%)
432.3 6 7.3
(42.1%)
67.6 6 7.5
(6.6%)
12.3 6 2.5
(1.2%)
267.1 6 6.9
(26.0%)
247.2 6 8.1
(24.1%)
446.8 6 19.5
(44.9%)
61.1 6 12.8
(6.1%)
15.8 6 2.1
(1.6%)
288.6 6 10.9
(29.0%)
183.6 6 11.6
(18.4%)
Notes: BER 5 basic excision repair; b-pol 5 polymerase-beta; Ape 5
apurinic/apyrimidinic endonuclease.
* Values are expressed as [Integrated Density Value (IDV)/cell (3103)] 6
SEM.
y
Percent of total chemiluminescence for a tissue calculated by: [IDV of
specific protein/Total IDV (5 proteins)] 3 100%.
INTANO ET AL.
208
Table 2. Proportional Abundances of BER Proteins in Nuclear Extracts
Prepared From Brain Obtained From Different Aged Mice
Protein
DNA Ligase I
DNA Ligase III
Xrcc1
b-pol
Ape/Ref1
3-Month-old mice 16-Month-old mice 28-Month-old mice
313.9 6 55.9*
(42.4%)y
86.4 6 12.5
(11.7%)
18.0 6 4.4
(2.4%)
211.1 6 37.6
(28.5%)
111.8 6 11.6
(15.0%)
288.9 6 37.6
(47.3%)
91.5 6 12.3
(14.9%)
21.8 6 12.9
(3.6%)
127.0 6 8.6
(20.8%)
81.6 6 5.1
(13.4%)
275.0 6 21.8
(59.1%)
73.1 6 6.3
(15.7%)
20.6 6 2.1
(4.4%)
58.0 6 6.8z
(12.5%)
38.4 6 6.9
(8.3%)
Notes: BER 5 basic excision repair; b-pol 5 polymerase-beta; Ape 5
apurinic/apyrimidinic endonuclease.
* Values are expressed as [Integrated Density Value (IDV)/Cell (3 103)]
6 SEM.
y
Percent of total chemiluminescence for a tissue calculated by: (IDV of
specific protein/Total IDV (5 proteins)) 3 100%.
z
Significantly different from 3-month-old value.
APE/REF-1, b-POL, or DNA ligase III to brain nuclear
extracts prepared from 3- and 28-m/o mice (Figure 4).
DISCUSSION
The potential role of DNA damage and mutagenesis in
aging has been controversial in part because, while there are
many studies showing an association between increased age
and increased genomic instability (4–10,12–14), there are
also studies that have not detected increased genomic instability with increased age (39,40). The decreased in vitro
UDG-BER activity observed in brain and liver nuclear extracts prepared from old animals in our study is consistent
with increased genomic instability with increased age and
with a recent study demonstrating reduced short-patch BER
in old mice compared with young adult mice (41).
UDG-BER activity varies among mouse tissues with
spermatogenic cell types exhibiting the highest level among
tested cell and tissues types followed by mitotically active
Sertoli cells, mitotically active thymocytes, small intestine, liver, and brain, but does not differ for a specific cell
or tissue type between young adult C57BL/6J, CD1, and
B6D2F1 mice (34). The present study demonstrates that UDGBER activity is higher in neonatal brain and liver than in
corresponding adult tissues. Only one difference has been
found between tested strains. BER activity in brain nuclear
extracts prepared from 8-d/o CD1 mice was lower than
samples obtained from 8-d/o B6D2F1 mice, but the overall
trend was similar, such that activity was highest in 6-d/o
mice and declined through 28-m/o.
A 50% reduction in UDG-BER activity has been
observed in spermatogenic cells obtained from old mice
compared with young mice and coincided with a 50%
reduction in the relative abundance of Ape (35). Addition
Figure 3. Uracil DNA glycosylase-initiated base excision repair (UDG-BER) activities for liver nuclear extracts prepared from 3-month-old (m/o) (white) and 28-m/o
male mice (black) to which increasing amounts of purified UDG (uracil DNA glycosylase) (A), Ape (apurinic/apyrimidinic endonuclease) (B), b-pol (polymerase b)
(C), and DNA ligase III (D) were added. Results are presented as means (6 SEM) of three replicate assays for each of three independent nuclear extract preparations.
SOMATIC BER DECLINES WITH AGE
209
Figure 4. Uracil DNA glycosylase base excision repair (UDG-BER) activities for brain nuclear extracts prepared from 3-month-old (m/o) (white) and 28-m/o male
mice (black) to which increasing amounts of purified UDG (uracil DNA glycosylase) (A), Ape (apurinic/apyrimidinic endonuclease) (B), b-pol (polymerase b) (C), and
DNA ligase III (D) were added. Results are presented as means (6 SEM) of three replicate assays for each of three independent nuclear extract preparations.
of purified APE/REF-1 to extracts prepared from old mice
restored UDG-BER activity (35). In contrast, addition of
purified udg or DNA ligase III elevated UDG-BER activity
in nuclear extracts prepared from young mice. These results
suggested that different enzyme activities limited UDGBER activity in samples prepared from young versus old
mice.
Unlike spermatogenic cell types, addition of single BER
proteins did not alter UDG-BER activity in brain or liver
nuclear extracts prepared from young or old mice. Thus,
these results for somatic tissues are very different from
results using spermatogenic cells and lead to the suggestion
that coordination and/or regulation of BER is different
between somatic and germline cells. The 80% reduction in
UDG-BER activity observed in brain nuclear extracts in the
current study coincided with reduced abundance of b-pol
and is consistent with data of Cabelof et al. (41). Rao et al.
(21) described reduced polymerase activity in neurons
obtained from old rats, which could be restored by the
addition of recombinant b-pol. The activities assessed by
Rao et al. (21) consisted of 39–59 exonuclease activity and
polymerase extension. BER activity was not examined
directly. In the present study, addition of purified b-POL did
not restore UDG-BER activity. Our results suggest that
although b-pol levels decrease with age, the reduced
abundance by itself is not a major factor limiting UDG-
BER activity. Cabelof et al. (41) also detected reduced b-pol
in liver samples prepared from old mice while our study did
not detect a change. The difference may be due to the different strains of mice used. Cabelof et al. used the inbred
C57BL strain, while we used a hybrid strain, B6D2F1.
The relatively constant abundances of other BER proteins
through the range of ages that were tested, combined with
the unaltered activity with the addition of specific purified
BER proteins, suggests that the abundance of any of the
individual BER proteins tested is not the mechanism by
which UDG-BER activity is limited in somatic tissues of
young mice, nor the mechanism by which activity is decreased relative to age in brain and liver. What then limits
UDG-BER activity in brain and liver nuclear extracts?
There are several possible explanations. (a) Perhaps Xrcc1,
not added to nuclear extracts in this study, is a limiting
protein. Xrcc1 RNA (42,43) and protein abundances
(Table 1) are extremely low in somatic tissues. (b) Failure
to detect limiting proteins in nuclear extracts could be
caused by the low abundances of BER proteins in the
extracts. Enhancement of BER via supplementation of
limiting proteins would stimulate the pathway only to the
extent that the remaining low abundance BER proteins
could accommodate the increase. (c) Extracts from old mice
might contain damaged and inactive BER proteins and
thereby mediate the decline in UDG-BER. This mechanism
210
INTANO ET AL.
could also explain why the addition of single BER proteins
does not restore UDG-BER activity in nuclear extracts of
somatic tissues obtained from old animals because more
than one protein would be involved in mediating the
decline. Supporting this hypothesis are results showing that
oxidatively damaged proteins accumulate with age (44) and
inactive b-pol accumulation has been observed with
increased age in neuronal extracts prepared from rats (20).
(d) Increased abundances of BER inhibitors might be found
in extracts prepared from old mice. (e) Finally, various
combinations of the above are possible.
In summary, this study has demonstrated that UDG-BER
activity declines in nuclear extracts prepared from brain and
liver in an age-related manner. The assay used interrogates
the ability of extracts to repair uracil in DNA via the shortpatch BER pathway. To better understand the possible
contribution of BER to increased genomic instability with increased age, repair of additional lesions must be examined,
and for somatic tissues, short- and long-patch BER must be
assessed.
ACKNOWLEDGMENTS
This publication was made possible by grant numbers ESO9136,
AG13560, AG14674, AG19360, and AG00205 from the National Institute
of Environmental Health Sciences and National Institute on Aging (NIA)
(National Institutes of Health [NIH]), the Environmental Hazards Center at
the South Texas Veteran’s Health Care System (STVHCS), the STVHCS,
the Nutritional and Interventional Gerontology Training Program, and
dissertation research support from the NIA. The contents are solely the
responsibility of the authors and do not necessarily represent the official
views of NIH or VHCS. We would like to thank Dr. Sam Wilson for
supplying antibody against DNA polymerase-b, Dr. Alan Tomkinson for
supplying purified DNA ligase I and IIIb and antibodies against DNA
ligase I and III, Ms. Terry Shadrock for professional assistance with the
references, and Dr. Rodney Levine for helpful discussions regarding the
interpretation of the data.
Address correspondence to Christi A. Walter, PhD, Department of
Cellular and Structural Biology, Mail Code 7762, The University of Texas
Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio,
TX 78229-3900. E-mail: [email protected]
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Received August 14, 2002
Accepted December 3, 2002
Decision Editor: James R. Smith, PhD