©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Action of Mitochondrial DNA Polymerase at Sites of Base Loss or Oxidative Damage (*)

Kevin G. Pinz , Shinya Shibutani , Daniel F. Bogenhagen (§)

From the (1) Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794-8651

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 . 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.


INTRODUCTION

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 APendonuclease (11) . However, understanding of the enzymology of mtDNA repair has lagged far behind studies of nuclear DNA repair.

One key enzyme in the response to damage to mtDNA is the mitochondrial DNA pol () . 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.

In this paper, we investigate the action of DNA pol 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 .


EXPERIMENTAL PROCEDURES

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.




RESULTS

Fig. 1 shows the design of an experiment intended to determine the action of DNA pol 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.

To determine the dNMP inserted by DNA pol 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.

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 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.''




DISCUSSION

DNA pol 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.

The tetrahydrofuran residue has two major effects on DNA replication by DNA pol . 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 exopolymerase 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.

Our results confirm that 8-oxo-dG has the potential to create frequent G 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 10base 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.


FOOTNOTES

*
This work was supported by Grant RO1-GM29681 from the National Institutes of Health and PO1-ES04068 from the NIEHS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 516-444-3068; Fax: 516-444-3218; E-mail: dan@pharm.sunysb.edu.

The abbreviations used are: AP, apurinic/apyrimidinic; pol, polymerase; 8-oxo-dG, 7,8-dihydro-8-oxo-2`-deoxyguanosine; DTT, dithiothreitol; HPLC, high performance liquid chromatography.

F. Ye and D. F. Bogenhagen, manuscript in preparation.

K. Pinz, unpublished observation.


ACKNOWLEDGEMENTS

We thank Robert Rieger and members of Francis Johnson's laboratory for synthesis of oligonucleotides.


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