From the
Mitochondrial DNA is subject to oxidative damage generating
7,8-dihydro-8-oxo-2`-deoxyguanosine (8-oxo-dG) residues and to
spontaneous or induced base loss generating abasic sites. Synthetic
oligonucleotides containing these lesions were prepared and used as
templates to determine their effects on the action of Xenopus
laevis DNA polymerase
Individual cells in higher eukaryotes contain several thousand
copies of the mtDNA genome
(1) . This genetic redundancy,
coupled with the rapid rate of mtDNA evolution
(2) and the
persistence of bulky damage in mtDNA
(3, 4, 5) ,
has led to the suggestion that damage to mtDNA is not commonly
repaired. However, some types of damage, such as spontaneous base loss
(6) and oxidative damage
(7) , are expected to be so
frequent that mitochondria must possess mechanisms for either repairing
the damage or permitting translesional synthesis during replication. As
expected, repair of some classes of damage to mtDNA has been documented
(8, 9) . Some mitochondrial enzymes involved in
recognition of DNA damage and its repair have been identified, such as
uracil DNA glycosylase
(10) and AP
One key enzyme
in the response to damage to mtDNA is the mitochondrial DNA pol
(
In this paper, we investigate the
action of DNA pol
Fig. 1 shows the design of an experiment intended to
determine the action of DNA pol
To determine the dNMP inserted by DNA pol
Substituting a tetrahydrofuran for the dG
residue in the template is expected to mimic the structure of a primer
opposite an apurinic site in DNA. The effects of the abasic site on
action of the pol
DNA pol
The
tetrahydrofuran residue has two major effects on DNA replication by DNA
pol
Our results confirm that 8-oxo-dG has the
potential to create frequent G
We thank Robert Rieger and members of Francis
Johnson's laboratory for synthesis of oligonucleotides.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
. An analogue of an abasic site in DNA,
tetrahydrofuran, was found to inhibit elongation by DNA polymerase
. When the DNA polymerase was able to complete translesional
synthesis, a dA residue was incorporated opposite the abasic site. In
contrast, elongation by DNA polymerase
was not inhibited by an
8-oxo-dG residue in the template strand. The polymerase inserted dA
opposite 8-oxo-dG in approximately 27% of the extended products. The
effects of these lesions on the 3`
5` exonuclease proofreading
activity of DNA polymerase
were also investigated. The 3`
5` exonuclease activity excised any of the four normal bases positioned
opposite either a tetrahydrofuran residue or 8-oxo-dG, suggesting that
proofreading may not play a major role in avoiding misincorporation at
abasic sites or 8-oxo-dG residues in the template. Thus, both of these
lesions have the prospect of causing high rates of mutation during
mtDNA replication.
endonuclease
(11) . However, understanding of the enzymology of mtDNA repair
has lagged far behind studies of nuclear DNA repair.
)
. This enzyme has only recently been purified from
higher eukaryotes
(12, 13, 14) . The intact
enzyme appears to contain a single catalytic subunit varying in size
from 125 to 145 kDa in different organisms. A similar variability in
predicted size has been observed for cloned DNA pol
from yeasts
(15) .(
)
DNA pol
contains an
associated 3`
5` exonuclease activity
(16, 17, 18, 19) and shows sequence
relationship to Escherichia coli DNA polymerase I. Since no
other DNA polymerase has been identified in mitochondria, it appears
likely that this enzyme must deal with DNA damage in the context of
both DNA replication and repair.
at sites of base loss and of oxidative damage
using site-specifically modified oligonucleotides with tetrahydrofuran
or 8-oxo-dG in the template strand. The effects of these lesions have
been studied for other DNA polymerases (see Refs. 20 and 21, and
references therein). In general, abasic sites provide an effective
kinetic block to elongation by DNA polymerases, although translesional
synthesis can occur, most frequently with incorporation of a dA residue
opposite the abasic site. In this study, we employ a synthetic
tetrahydrofuran residue as an analogue of a natural abasic site
(22) . This analogue has been used widely in previous studies of
the action of other DNA polymerases at abasic sites. As a model for
oxidative damage in DNA, we have used 8-oxo-dG. The incidence of
8-oxo-dG in DNA and its mutagenic consequences have been reviewed
(23) . DNA polymerases from a variety of sources read through
8-oxo-dG residues efficiently but show variable frequencies of
misincorporation of dA opposite the adduct
(20) . Oxidative
damage producing 8-oxo-dG has been reported to occur in mtDNA at a
frequency approximately 10 times greater than in nuclear DNA
(7) , possibly as a consequence of the high rates of oxidative
metabolism in mitochondria. Hayakawa et al. (24) have
reported a dramatic increase in the occurrence of 8-oxo-dG in mtDNA in
mouse liver following treatment with azidothymidine. Thus, it is
particularly important to determine the effect of this lesion on
replication by DNA pol
.
Materials
Mature Xenopus laevis females
were obtained from Xenopus I (Ann Arbor, MI). The Sep-Pak C18
cartridges and the Gen-Pak FAX anion exchange column were obtained from
Millipore (Bedford, MA). Other prepacked columns, chromatography
matrices, and nucleotides were obtained from Pharmacia Biotech Inc.
Protease inhibitors leupeptin, aprotinin, and E-64 were obtained from
Boehringer Mannheim. Benzamidine HCl, dithiothreitol (DTT), bovine
serum albumin, aphidicolin, and pepstatin were obtained from Sigma.
HaeIII restriction endonuclease and polynucleotide kinase were
obtained from New England Biolabs (Beverly, MA). Radioisotopes were
obtained from ICN Radiochemicals (Irvine, CA). Unmodified synthetic
oligodeoxynucleotides were prepared by standard automated solid state
techniques. Oligodeoxynucleotides containing tetrahydrofuran or
8-oxo-dG residues were prepared as described by Takeshita et al. (22) and by Bodepudi et al. (25) ,
respectively. All oligodeoxynucleotides were purified by HPLC
chromatography.
DNA pol
DNA pol was purified from
Triton X-100 lysates of X. laevis ovary mitochondria by a
modification of the procedure of Insdorf and Bogenhagen
(13) .
Following the hydrophobic interaction chromatography step in the
published procedure, the enzyme was applied to a HiLoad 16/60 Superdex
200 gel filtration column in buffer containing 5% glycerol, 300
m
M NaCl, 20 m
M Tris (pH 8), 2 m
M DTT, 1
m
M MgCl
, 0.02% Triton X-100, 2 m
M
benzamidine HCl, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1
µ
M pepstatin A, 1 µ
M E-64, and 100 µg/ml
gelatin. DNA polymerase activity was detected in assays using
poly(rA)
oligo(dT) template as described
(13) . Fractions
containing the peak of activity were applied to a 1-ml
heparin-Sepharose (HiTrap; Pharmacia) column in buffer containing 5%
glycerol, 100 m
M NaCl, 20 m
M Tris (pH 8), 2
m
M DTT, 1 m
M EDTA, 0.02% Triton X-100, 2 m
M
benzamidine HCl, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1
µ
M pepstatin A, 1 µ
M E-64, and 100 µg/ml
gelatin. DNA pol
was eluted with a gradient of increasing KCl.
DNA pol
purified by this procedure had a specific activity
comparable to that reported for a more lengthy purification scheme
(13) but was found to contain a higher proportion of intact
140-kDa enzyme by SDS-PAGE analysis. The DNA pol
activity on
activated DNA and poly(dA)
oligo(dT) substrates retained 95 and 88%
of activity in the presence of 1 µ
M ddTTP and 20 µg/ml
aphidicolin, respectively.
Polymerase Reactions
Oligonucleotide substrates
were prepared as follows; 10-20 pmol quantities of 11-mer or
13-mer primers (sequences shown in Fig. 1) were 5` labeled using
polynucleotide kinase and [-
P]ATP.
Oligonucleotides were precipitated with ethanol in the presence of 10
µg of glycogen carrier and purified by electrophoresis on 20%
polyacrylamide gels containing 8
M urea and recovered by crush
elution and chromatography on Sep-Pak C18 cartridges. Labeled
oligonucleotides were annealed with a 1.5- or 2-fold excess of 31-mer
oligonucleotide containing either dG, tetrahydrofuran, or 8-oxo-dG
(sequences shown in Fig. 1) in 10 m
M Tris, pH 8.4, 50
m
M NaCl, 2 m
M MgCl
. Polymerase reactions
contained 3.5 pmol of oligonucleotide, 200 µ
M of each
dNTP, 1 µg of bovine serum albumin, 500 µ
M DTT, 10
m
M MgCl
, 40 m
M KCl, 20 m
M Tris,
pH 8.4, and 300 units of DNA pol
in a volume of 50 µl.
Extension reactions were continued for 20 min at 25 °C and
terminated by incubation at 65 °C for 5 min. Reactions containing
fully extended products were digested with HaeIII restriction
endonuclease for 90 min at 37 °C. All reaction mixtures were
precipitated with ethanol in the presence of 10 µg of glycogen
carrier, analyzed by electrophoresis on gels containing 20%
polyacrylamide with 8
M urea, and detected with
autoradiography. In the experiment shown in Fig. 3, the
HaeIII digestions products were repurified by chromatography
on a Gen-Pak FAX anion exchange column and analyzed by electrophoresis
using the two-phased (15
72
0.04 cm) gel system of
Shibutani
(26) containing 7
M urea only in the top 10
cm of the gel. For the exonuclease assays, dNTPs, bovine serum albumin,
and DTT were omitted, and 40 units of DNA pol
were assayed.
Reactions were terminated by placing 3-µl aliquots removed at
various times from the 20-µl reaction mixtures into formamide DNA
loading solution (90% formamide, 20 m
M Tris (pH 8), 25
m
M EDTA, 50 µg/ml bromphenol blue, 50 µg/ml xylene
cyanol). All aliquots were electrophoresed on 20% polyacryamide gels
containing 8
M urea and the radioactivities of substrates and
products on the gel were quantified using a Betascope 603 isotope
detector (BetaGen Corp., Framingham, MA).
Figure 1:
Oligonucleotide templates to test
effects of tetrahydrofuran and 8-oxo-dG on polymerase and exonuclease
activities of DNA pol . Panel A, polymerase assay. A
5`
P-labeled (
) primer is annealed to a
template containing a dG, a tetrahydrofuran, or an 8-oxo-dG residue at
position X. DNA pol
may extend the primer beyond the
lesion but will be expected to leave a heterogeneous
(``frayed'') 3` end due to its associated 3`
5`
exonuclease activity. The extended strands can be cut with
HaeIII to eliminate this 3` end heterogeneity. The identity of
the nucleotide inserted opposite X by DNA pol
is
determined as described by Shibutani (26). Panel B,
exonuclease assay. A series of 5`-
P-labeled (*)
13-nucleotide primer strands with each of the four deoxynucleotides at
the 3` position are annealed separately to a complementary strand
containing a dG, a tetrahydrofuran, or an 8-oxo-dG residue at position
X. These duplexes are incubated with DNA pol
in the
absence of dNTPs and analyzed by electrophoresis to determine the
kinetics of removal of the 3` terminal residue by the exonuclease
activity of the polymerase.
Figure 3:
dNMP incorporation opposite abasic sites
and 8-oxo-dG residues. The HaeIII-cut products shown in Fig. 2
( lanes A3, B3, and C3) were
HPLC-purified and subjected to electrophoresis in lanes
1-3, respectively, on a two-phased polyacrylamide gel as
described by Shibutani (26). Markers ( lanes M)
consisted of a mixture of 18-mers containing A, C, G, or T at the
position opposite the lesion ( X) in the sequence shown in Fig.
1 and 17-mer and 16-mer oligonucleotides containing 1- or 2-nucleotide
deletions at this site.
at abasic sites or 8-oxo-dG
residues in oligodeoxynucleotide templates. This assay is essentially
as described
(26) with the addition of a step to recut the
products with HaeIII endonuclease to eliminate 3` terminal
heterogeneity expected due to the exonuclease associated with DNA pol
. The results of this polymerase assay are shown in Fig. 2. As can
be seen in lanes A2 and B2, a significant
fraction of the primer was extended under our polymerase reaction
conditions when the template contained a dG residue (control) or
8-oxo-dG. In contrast, only a low level of translesional synthesis was
observed on a template containing the tetrahydrofuran residue ( lane C2). A 10-fold longer autoradiographic exposure was
required to detect bypass replication ( lane set C`). We
conclude that an abasic site is an effective barrier to elongation by
DNA pol
. We considered the possibility that action of an AP
endonuclease to cleave the template at the tetrahydrofuran residue
might contribute to premature termination at the abasic site. However,
control experiments showed that the template strand was not incised
during the course of our reactions.
(
)
As shown in
Fig. 1
, the 3` end of the primer is located one nucleotide away
from the abasic site in the template strand. Most extension products
obtained with the tetrahydrofuran template showed addition of only a
single residue, although further extension to incorporate a residue
opposite the abasic site was readily detected as a 15-mer in lanes C2 and C3. This suggests that both incorporation
opposite the tetrahydrofuran residue and extension of a primer with a
3` terminus ``paired'' with the abasic site are unfavorable
reactions.
opposite
an 8-oxo-dG or tetrahydrofuran residue, the fully extended products
were digested with HaeIII endonuclease as shown in lanes 3 of Fig. 2. The resulting 18-mers were recovered
by HPLC and subjected to electrophoresis using a two-phased gel system
(26) . The autoradiogram in Fig. 3 showed that dC, the correct
base, was incorporated in 73% of fully extended products, while dA was
misincorporated opposite 8-oxo-dG in approximately 27% of the products.
Fig. 3
also shows the products of replication past the abasic
site analogue. The large majority of these bypass replication products
contained dA residues opposite the abasic site, as reported for other
DNA polymerases
(21) . Thus, abasic sites in mtDNA can have two
sorts of serious consequences. First, as shown in Fig. 2, these
lesions can serve as blocks to replication. Second, the low frequency
of successful bypass replication through abasic sites by DNA pol
is expected to be highly mutagenic.
Figure 2:
Products of extension by DNA pol
through damaged sites. The polymerase assay was conducted as outlined
in Fig. 1 using template strands containing dG ( lane set A), 8-oxo-dG ( set B), or
tetrahydrofuran ( set C). In each set, the
5`-
P-labeled primer is shown in lane 1,
and the heterogeneous extension products are shown in lane 2. Cleavage of the extension products with
HaeIII generated the discrete product shown in lane 3. The right hand panel containing lanes C` shows a 10-fold longer autoradiographic exposure of
the lanes in set C to detect the low levels of
extension past the tetrahydrofuran residue.
The misincorporation of dA
residues opposite 8-oxo-dG or an abasic site is the net result of the
action of two enzymatic centers in the DNA polymerase, the polymerase
domain and the 3` 5` exonuclease. Foury and Vanderstraeten
(27) have shown recently that the exonuclease domain of the
Saccharomyces cerevisiae DNA pol
plays an important role
in increasing the fidelity of replication. In this study, we used a
variety of oligodeoxynucleotide substrates to investigate the action of
the proofreading exonuclease on templates containing damaged residues.
The basic approach is diagrammed in Fig. 1. We used a set of four
13-mer primers with identical sequences, except for the 3` terminal
residue. The primers anneal to the template with their 3` ends
positioned opposite a dG residue (undamaged control), an abasic site or
8-oxo-dG. We have previously shown that the 3`
5` exonuclease of
X. laevis DNA pol
excises a 3` terminal mismatch more
rapidly than a 3` base paired residue, as expected for a proofreading
activity
(16) . Fig. 4 A extends this analysis to
examine the activity of the exonuclease on each of the four potential
3` terminal nucleotides opposite a dG residue. In this control series,
the G:C pair shows the greatest stability to exonuclease, although even
the paired 3` terminus is subject to exonucleolytic attack. G:G and G:A
mismatches are excised much more rapidly, while the G:T mismatch is
excised at an intermediate rate. Similar results have been observed by
Longley and Mosbaugh
(19) for the porcine DNA pol
. In
both instances, the specificity of the exonuclease for a mismatched 3`
terminus is not absolute.
exonuclease are shown in
Fig. 4B. All four primers with 3` ends positioned
opposite the abasic site were actively attacked by the 3`
5`
exonuclease. The dA 3` terminus is not particularly stable to the
action of the exonuclease in comparison with other 3` residues. The
influence of 8-oxo-dG on the 3`
5` exonuclease of pol
are
shown in Fig. 4 C. All four primer 3` ends are recognized
as inappropriate by the enzyme in this context and are actively
attacked. A 3` terminal dC residue opposite 8-oxo-dG is removed at the
lowest rate, although this pair is not as stable to exonucleolytic
attack as the dC:dG pair (Fig. 4, compare A and
C). This is consistent with the observation that dC is
incorporated in most extension events (Fig. 3). Interestingly, an
8-oxodG:dA 3` terminus is not particularly stabilized against
exonucleolytic attack.
Figure 4:
Contribution of the pol exonuclease
to mutagenesis at damaged residues. 5`-
P-labeled 13-mer
oligodeoxynucleotides containing G, A, T, or C at the 3` end were
annealed to a 1.5-fold excess of templates containing dG ( panel A), tetrahydrofuran ( panel B), or
8-oxo-dG ( panel C) opposite the 3` end of the primer,
as diagrammed in Fig. 1 B. Duplexes were incubated with pol
for varied times under polymerase buffer conditions in the
absence of deoxynucleoside triphosphates to monitor exonuclease
activity. The labeled 13-mer substrate and 12-mer products of the 3`
5` exonuclease were quantified by isotopic scanning as described
under ``Experimental Procedures.''
is the polymerase responsible for replication
and, most likely, repair of mtDNA. This polymerase is known to be
highly processive
(28) and to replicate DNA with high fidelity
(17) . Single point mutations have been implicated in the
etiology of a number of genetic diseases caused by mutations in mtDNA
(reviewed in Ref. 29). Recently, evidence has been accumulating to
support the view that damage to mtDNA may contribute to aging and to
other degenerative diseases that may have a multifactorial etiology
(reviewed in Ref. 30). Given the growing literature on mtDNA mutations
and disease, we set out to study the effects of two common sorts of DNA
template damage on the action of the mitochondrial DNA pol
. We
found that abasic sites and 8-oxo-dG residues have disparate effects on
the action of the polymerase, although both lesions have a potential to
be highly mutagenic in vivo.
Effects of an Abasic Site
The abasic site analogue,
tetrahydrofuran, was used in these experiments for two reasons. First,
it differs from an authentic abasic site only by the replacement of an
hydroxyl group on the 1` carbon of deoxyribose with a hydrogen, so that
it is a structurally accurate analogue of an abasic site. Second, this
residue can be incorporated at precise positions during automated
synthesis of oligonucleotides. Although the greater chemical stability
of the tetrahydrofuran residue suppresses action of AP lyases and can
influence the detailed pathway of abasic site repair
(31) ,
these features are not relevant to the present study.
. First, it greatly inhibits the frequency of translesional
synthesis. In a series of experiments conducted under a variety of
solution conditions we observed an average of 80% blockage of
replication at the abasic site.
Second, when the polymerase
is able to bypass the lesion, it virtually always incorporates dA
opposite the abasic site. If an abasic site in vivo is
generated by loss of a base other than thymine the incorporation of a
dA residue will be mutagenic. These results are similar to those
observed for other DNA polymerases
(21) . In particular, it is
interesting to note that the presence of a proofreading exonuclease in
T4 phage DNA polymerase has been observed to decrease the ability of
the enzyme to bypass an abasic site
(21) . The exo
polymerase may engage in multiple rounds of dAMP incorporation
and excision opposite the abasic site in a dAMP turnover reaction that
produces a kinetic block to further elongation. Thus, mutagenesis of
the 3`
5` exonuclease site or selective inhibition of the
exonuclease activity of DNA pol
might increase the frequency of
elongation past abasic sites. It is also possible that mitochondria may
contain associated replication factors that might influence the
efficiency of bypass synthesis through abasic sites. The search for
such an activity is encouraged by the observation that proliferating
cell nuclear antigen can increase translesional synthesis by DNA pol
on a template containing a DNA photoadduct
(32) .
Effects of 8-Oxo-dG
Shibutani et al. (20) have shown that DNA polymerases from different sources vary
considerably in their frequency of misincorporation of dA opposite
8-oxo-dG in DNA. For example, DNA polymerases and
exhibited
a high frequency of incorporation of dA opposite 8-oxo-dG, while DNA
pol
showed minimal misincorporation in the same context.
Misincorporation of dA is thought to reflect the propensity of 8-oxo-dG
to adopt a syn conformation about the glycosidic bond to allow
base pairing with dA
(23, 33) . In the present study, we
found that 8-oxo-dG induces a moderately high rate of incorporation of
dA opposite the G adduct during replication by DNA pol
. It would
appear that a primer-template with 3`-dA in the primer positioned
opposite 8-oxo-dG provides the polymerase with a suitable substrate for
elongation. The results shown in Fig. 4 C indicate that a
dA:8-oxo-dG 3` pair is not as resistant to attack by the 3`
5`
exonuclease center of the polymerase as a Watson-Crick dC:dG base pair.
Thus, preferential misincorporation of dA opposite 8-oxo-dG principally
reflects the specificity of the polymerase domain of the enzyme. It
would be interesting to determine the rate of dA misincorporation by
DNA polymerase
in the absence of an active 3`
5`
exonuclease domain.
T transversion mutations in
mtDNA. The biological importance of this lesion then depends on the
actual incidence of 8-oxo-dG in mtDNA and the rate of its repair. It
has been suggested that 8-oxo-dG could be generated at an increased
rate in mtDNA by superoxide radicals produced as a byproduct of
oxidative metabolism
(34) . However, the reported frequency of
8-oxo-dG in mtDNA varies widely in different studies. Richter et
al. (7) originally reported a high steady state abundance
of 1 8-oxo-dG residue/8000 bases in mtDNA. In contrast, a recent
investigation of the incidence of 8-oxo-dG in mtDNA in which the
oxidized sites were probed as FAPY (formaminopyrimidine)-glycosylase
sensitive sites observed less than 1 lesion/5
10
base pairs
(35) . The extent to which differences in
methodology, tissue sources, or the rate of repair of oxidative damage
(9) contribute to this discrepancy remains to be established.
One report of a massive conversion of dG to 8-oxo-dG in mtDNA following
administration of azidothymidine
(24) has not, to our
knowledge, been reproduced in other studies. While our experiments have
confirmed that 8-oxo-dG induces frequent misincorporation by DNA pol
, the biological significance of this lesion in mtDNA is not yet
completely understood.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.