Cardnogenesb vol.13 no. 11 pp. 1967-1973, 1992

Repair of mitochondrial DNA after various types of DNA damage in Chinese hamster ovary cells

Susan P.LeDoux, Glenn L.Wilson, Edward J.Beecham1, Tinna Stevnsner1, Karsten Wassermann1 and Vilhelm A.Bohr1'2

2

To whom correspondence should be addressed

Using methodology recently developed to assess gene-specific DNA repair, we have demonstrated that it is possible not only to study mitochondrial DNA repair, but also directly to compare mitochondrial and nuclear DNA repair in the same biological sample. Complex enzymatic mechanisms recognize and repair nuclear DNA damage, but it has long been thought that there was no DNA repair in mitochondria. Therefore, in an attempt to delineate more clearly which DNA repair mechanisms, if any, are functioning in mitochondria, we have investigated the repair of several specific DNA lesions in mitochondrial DNA. They include cyclobutane dimers, cisplatin intrastrand adducts, cisplatin interstrand crosslinks and alkali-labile sites. We find that pyrimidine dimers and complex alkylatkm damage are not repaired in mitochondria] DNA, and that there is minimal repair of cisplatin intrastrand crosslinks. In contrast, there is efficient repair of cisplatin interstrand crosslinks as evidenced by ~ 70% of the lesions being removed by 24 h. Additionally, there is efficient repair of jV-methylpurines following exposure to methylnitrosourea with ~ 70% of the lesions being removed by 24 h. The results of these studies reveal that repair capacity of mitochondrial DNA damage depends upon the type of lesion produced by the damaging agent. We speculate that a process similar to the base excision mechanism for nuclear DNA exists for mitochondrial DNA but that there is no nucleotide excision repair mechanism to remove more bulky lesions in this organelle.

Introduction Human mitochondria have their own DNA, which is doublestranded, circular and contains —16 569 base pairs. The mitochondrial genome has been sequenced in its entirety and consists of genes that code for 13 subunits of the respiratory chain complexes, 22 tRNAs and two rRNAs (1). Until recently, the only mitochondrial DNA defect that appeared te beof significance was that of a mutation in one of the mitochondrial ribosomal RNA genes which conferred chloramphenicol resistance (2). However, within the past three years defects in die mitochondrial genome have been described in a number of human diseases. The initial description by Holt et al. (3) of deleted mitochondrial DNA in patients with mitochondrial diseases has been confirmed by 'Abbreviations: MNU, methylnitrosourea; DHFR, dihydrofolate reductase; CHO, Chinese hamster ovary; TE buffer, 10 mM Tris-HCl, 1 mM EDTA; CP buffer, 50 mM citrate phosphate buffer, pH 7.0.

1967

Downloaded from http://carcin.oxfordjournals.org/ at Tulane University Library, Serials Acquisitions Dept. on January 13, 2015

Department of Structural and Cellular Biology, University of South Alabama, Mobile, AL 36688 and 'Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

numerous studies which found that mitochondrial deletions occurred in >90% of cases with Kearns—Say re syndrome (4). Additionally, mitochondrial point mutations have been associated with Leber's hereditary optic neuropathy (5), Pearson's syndrome (6) and some cases of chronic progressive external ophthalmoplegia (5). Accumulations of mutations in mitochondrial DNA have also been reported in the brains of patients with Parkinsonism (7). These findings suggest that mitochondrial DNA defects are of biological importance. DNA repair processes play a crucial role in the susceptibility of cells to the effects of mutagens. The importance of these processes is exemplified in the human disorder xeroderma pigmentosum, where a DNA repair defect has been documented and where patients are extremely sensitive to the mutagenic effects of a variety of agents. DNA repair processes also are important in cancer chemotherapy. Malignant cells often demonstrate an increased rate of cell division and DNA replication, thereby increasing their sensitivity to the cytotoxic effects of agents that either damage DNA directly or inhibit DNA repair processes. In attempts to understand the mechanisms involved in these processes, much attention has focused on DNA repair processes in the nuclear genome. Complex enzymatic mechanisms for recognition and repair of nuclear DNA damage have been demonstrated with specific pathways being involved in the repair of different lesions. Similar pathways have not been demonstrated for mitochondrial DNA even though mitochondrial DNA is extremely sensitive to DNA damage. It has been widely assumed that there was no DNA repair capacity in mitochondria. In fact, only recently has evidence begun to accumulate which suggests that there may be some capacity for excision repair in mitochondria (8-15). The study of mitochondrial DNA repair processes has been impaired greatly by the difficulty in isolating significant quantities of mitochondrial DNA free of nuclear DNA contamination. However, using quantitative Southern analysis with a probe specific for the entire mitochondrial DNA sequence we have shown that it is possible to study repair selectively within the mitochondrial genome (15). This methodology is based on the formation of single-strand breaks at the site of the lesion to be investigated and then quantitation of the formation and removal of these strand breaks in the DNA fragment of choice. Thus, one can assess DNA damage and repair in the mitochondrial genome using a mitochondrial DNA probe and evaluate repair in the nuclear genome with a probe for a nuclear sequence on the same blot using the same biological sample. In an attempt to delineate more clearly which repair pathways are functional in the mitochondrion, the present studies were designed to establish whedier there is repair of the mitochondrial DNA in hamster cells following exposure to different DNAdamaging agents that produce different types of lesions. Exposure of cells to LTV irradiation is the most extensively used model for investigating DNA damage and repair. Its relevance is apparent since most living organisms must contend with the genotoxic effects of solar LTV irradiation. The predominant lesion formed is the cyclobutane pyrimidine dimer. The formation and repair

S.P.LeDoux et al.

We have compared the repair of each type of lesion in mitochondrial DNA with that in bulk DNA and with that in a specific nuclear sequence which codes for a portion of the transcriptionally active dihydrofolate reductase (DHFR) gene. Our results demonstrate that repair of mitochondrial DNA damage depends upon the type of modification. In the mitochondria, there is efficient repair of certain types of lesions and little or no repair of others. Materials and methods Cell culture Chinese hamster ovary (CHO) Bl 1 cells (16) were grown in Ham's F-12 medium (without glycine, hypoxanthine and thymidine; GBCO 78—5474) supplemented with 500 nM methotrexate, 10% dialyzed calf serum and gentamicin. Forty-eight hours before DNA-damaging agents were used, 5 x lO^cells were seeded per 10 cm dish in culture medium containing 0.2 /iCi/ml [3H]thymidine and 10 M thymidine to label the cells uniformly. Prior to treatment with the DNA-damaging agent, the medium was withdrawn and the cultures were rinsed with PBS or Hanks' Balanced Salt Solution. The cultures were then exposed to the damaging agent. The procedures for exposure to the individual agents have been described previously: MNU (17), nitrogen mustard (18), UV light (19) and cisplatin (20). The doses used in these experiments are equal to or less than those that have been described previously for the study of gene-specific repair of nuclear DNA damage in these cells (17-20). Because of the increased methylation of mitochondrial DNA, the dose of MNU used was one-half of that used in the repair studies of the overall genome or DHFR gene. After treatment with the damaging agent, the cultures were either prepared immediately for assay of DNA damage or replenished with repair medium (Ham's F-12, 10% dialyzed calf serum, gentamicin, 10~3M bromodeoxyuridine, and 10~6M fluorodeoxyuridine). Cultures containing repair medium were assayed for DNA damage after 8 or 24 h of incubation. Each repair experiment was performed at least in duplicate. Repair assay for gene-specific DNA damage This assay was adapted from that described in detail elsewhere (21). Briefly, cells were lysed in a solution composed of 10 mM Tris-HCl, 1 mM EDTA, 0.5% sodium dodecyl sulfate and incubated for 12 h at 37°C in proteinase K (0.1 mg/ml) (Sigma). The DNA was extracted with an equal volume of phenol, phenol/chloroform (1:1) and chloroform. It was precipitated with ethanol, resuspended in TE buffer (Tris-HCl, 1 mM EDTA) or CP buffer (50 mM citrate phosphate buffer, pH 7.0), and quantitated by determining absorbance at 260 nm. Subsequently, the DNA was treated with the endonuclease Kpn\ (10 units/fig DNA for 6 - 1 2 h at 37°C), and completeness of the digestion was verified on minigels. Then samples were centrifuged on CsCl gradients. The fractions containing parental DNA were identified by scintillation spectrometry, pooled, dialyzed and ethanol precipitated. The samples were resuspended in TE or CP

1968

buffer. The procedures used to form single-strand breaks in the DNA at the site of the lesion varied with the different agents. Following exposure to 20 J/m2 UV light, pyrimidine dimers were detected by treatment with T4 endonuclease V as previously described in detail (19). Intrastrand adducts in DNA from cells exposed to 300 /»M cisplatin were analyzed using UvrABC endonuclease as previously described (22). Cisplatin interstrand crosslinks were detected using a denaturation-renaturanoo procedure, modified after Vos and Hanawalt (20). After the denaturation-renaturation treatment cross-linked DNA readily anneals while the non-cross-linked DNA remains single-stranded. Briefly, 1 jig of DNA from cisplatin-treated cells was denatured with 30 mM NaOH in 20 y.\ for 20 min at 37°C and then kept on ice. The DNA samples were mixed with 10 x loading buffer and dye for a final concentration of 0.26% Rcoll, 0.1 mM EDTA, and 0.0025% bromocresol green and loaded on a 0.5% neutral agarose gel buffered in 40 mM Tris acetate and 2 mM EDTA. Non-denatured samples were also loaded as controls. Electrophoresis was carried out at 26 V for 16—20 h. For detection of alkali-labile sites following depurination of DNA treated in vivo with MNU or nitrogen mustard, the samples were heated at 70°C for 30 min in TE or CP buffer, pH 7.0, to depurinate DNA followed by an incubation in 0.1 N NaOH at 37°C for alkaline hydrolysis of apurinic sites (17,18). To ensure accuracy in these experiments, extreme care was taken in the quantitation of these samples so that 5 \t% of DNA was loaded in each lane. The samples were prepared in duplicate and were quantitatively loaded onto the gel. Initially an internal standard (linearized pBR322 at 0.05 ng/sample) was added to the samples (similar to that shown in Figures 3 and 5). However, with refinement of the assay it was determined that a more discriminating measure of quantitative loading of the gel was provided by densitometric scanning of the negatives from the pictures taken of the ethidium bromide stained gels. For this reason, the inclusion of an internal standard was discontinued. Electrophoresis was performed using 0.6% alkaline agarose gels in a buffer consisting of 30 mM NaOH, 1 mM EDTA for 20 h at 30 V. Southern transfer and hybridization were performed as described in detail previously (21). Quantitation of the hybridization was done by scanning densitometry of the autoradiograph. The fraction of fragments free of damage (zero class) was determined by dividing the band intensity of the treated sample by the intensity of the non-treated sample. The number of lesions per fragment was then determined using the Poisson expression (s = —In Po; where s is the number of lesions per fragment and Po is the fraction of fragments free of damage) under the assumption that damage is random within a given restriction fragment and that the repair of that fragment is non-processive. The break frequency for the 0 h repair data was used as the initial break frequency. The repair efficiency was then calculated as the initial break frequency minus the break frequency after repair divided by the initial break frequency. For these experiments, a repair efficiency of > 15% was considered to be above the background noise range of the assay and therefore significant. Analysis of DNA distribution on CsCl gradients To demonstrate that parental and replicated mhcchondrial DNA segregate in CsCl gradients in a manner which is similar to that of nuclear DNA, 100 jig of restricted cellular DNA was run on CsCl gradients. The gradients were collected and 50 li\ aliquots of the individual fractions were denatured in 0.25 M NaOH and slot blotted onto a nylon support membrane. The filter was probed for mitochondrial DNA. DNA probes The probe utilized in hybridizations for the mitochondrial sequence was a gift from Dr Allen Neims (University of Florida, Gainesville, FL) and consists of the entire 16.5 kb mouse mitochondrial genome inserted at the Sad she of pSP64. This probe recognizes a 16 kb fragment when hybridized to CHO DNA digested with Kpnl. The probe utilized in hybridizations for the sequence containing the DHFR gene was the pMB5 plasmkl previously described (19). This probe recognizes a 14 kb fragment of Apnl-restncted CHO DNA which contains the 5' half of the DHFR gene. The probes were labeled using a nick translation kit from BRL (Bethesda Research Laboratories) following the protocol described by the manufacturer. Specific radioactivity of the resultant hybridization probes was usually — 109 c.p.m.//tg of DNA. The hybridization mixture contained 1-2X10 7 c.p.m. of probe.

Results Replication A critical step in these experiments is the incorporation of the heavy thymidine analog bromodeoxyuridine into replicating DNA during the repair period. This allows for the separation of repaired parental DNA from any newly replicated DNA and assures that the phenomenon being studied is a repair process and not resynthesis of new mitochondrial DNA during the repair

Downloaded from http://carcin.oxfordjournals.org/ at Tulane University Library, Serials Acquisitions Dept. on January 13, 2015

of pyrimidine dimers can be assessed using the pyrimidine dimerspecific T4 endonuclease V to create strand breaks at sites of the dimers. A second DNA-damaging agent that we have used in these studies is the cancer chemotherapeutic drug cisplatin. The majority of DNA lesions formed by this compound (70—90% of total) are the intrastrand adducts between adjacent purines. This adduct can be detected with the bacterial enzyme UvrABC endonuclease, which nicks the DNA at the site of the lesion. A rare, but important cisplatin-induced lesion is the interstrand crosslink. We can detect this lesion at the gene level by use of denaturation—reannealing gel electrophoresis and quantitative Southern blotting. The third group of DNA-damaging agents investigated here are alkylating agents. These compounds are important cancer chemotherapeutics and are also among the environmental DNA-damaging toxins. For these studies, we examined repair in the mitochondrial DNA after treatment of the cells with methylnitrosurea (MNU*) and the nitrogen mustard bis(2-chloroethylmethyl)amine. MNU interacts with DNA to form methylated bases, while nitrogen mustard reacts with DNA to form more complex alkylation products. For both of these alkylating agents, the predominant lesions formed are Nalkylpurines, which we can detect in our assay by a direct chemical reaction in which the strand breaks are generated by neutral heat depurination followed by alkaline hydrolysis.

Repair of mitochondrial DNA

Initial damage Inherent in this methodology is the determination of initial break frequencies. Table I shows the level of damage in the mitochondrial DNA for the different lesions investigated. For

a comparison, this table also includes previously published data for the formation of damage in the endogenous, nuclear DHFR gene. A significant increase in damage in mitochondrial DNA compared to the DHFR gene was only seen when the damaging agent was MNU, and not for any of the other lesions. The frequency of lesions introduced with MNU in the mitochondrial DNA was as high as that introduced in the DHFR gene at a 2-fold higher dose. With MNU the increase in alkylation damage in mitochondrial DNA resulted in an inability to determine a 'zero class' value so that the dose of MNU used in studies of repair in the nuclear sequence containing the DHFR gene had to be reduced by one-half in order for the initial break frequency to be between 1 and 2 breaks per fragment—the value that allows for the best assessment of repair efficiency. Repair Analysis of repair of UV pyrimidine dimers in the mitochondrial genome of CHO cells is shown in Figure 2 and Table I. For these experiments, CHO cells were exposed to 20 J/m2 UV light (254 run) and then lysed immediately or allowed 8 or 24 h to repair. Samples of parental DNA were either treated or not treated with T4 Endonuclease V, electrophoresed, transferred to. a nylon support membrane and probed. From the resulting autoradiograph (Figure 2) the break frequency within the mitochondria] genome was determined at 0, 8 and 24 h. No repair (~ 10% or less) of Table I. Damage and repair in the mitochondrial genome 24 h

8h

Oh

Mitoch.

DHFR Mitoch. Mitoch. Lesions Lesions Lesions

IS

0 HR

24 HR

to

!•

K

M

MNU, 1 mM MNU, 0.5 mM Nitrogen mustard Cisplatin ICL Cisplatin LA UV

30

6 12 18 24 30 36 6 12 18 24 30 36

Fig. 1. Analysis of DNA distribution on CsCl gradients. Cells were grown in the presence of BrdUrd for 0 or 24 h. One hundred mkrograms of restricted cellular DNA was run on CsCl gradients. Thirty-eight fractions were collected beginning at the top of gradients so that high density DNA is to the right in this graph. Aliquots (50 /J) of the individual fractions were blotted onto a nylon membrane. The slot-Wot apparatus was loaded from left to right with six samples per horizontal row. The bound DNA was hybridized with the 32P-labeled mitochondrial DNA probe.

%repair

Lesions % repair

12 6 51 18 14

0.38 0.63 0.15 1.13 1.23

1.5 1.19 0.63 0.56 1.38 1.38

0.93 1.0 1.64 1.14

1.04 0.59 0.28 1.13 1.18

68 0 73 18 11

Lesions are given as frequency per 10 kb. ICL, interstrand crosslink; IA, intrastrand adduct

TIME Hrs ENDOV

- +

24

8

0

-

+

- + 23.1 9.4

6.6

Fig. 2. Repair analysis of pyrimidine dimers within the mitochondrial genome of CHO cells. CHO cells were exposed to 20 J/m2 UV light (254 nm) and then lysed immediately or allowed to repair for 8 or 24 h. High mol. wt DNA was isolated and digested to completion with Kpnl. Samples were either treated (+) or not treated ( - ) with T4 endonuclease V. The first and second lanes are from 0 h repair cultures. The third and fourth lanes are from 8 h repair cultures. The fifth and sixth lanes are from 24 h repair cultures.

1969

Downloaded from http://carcin.oxfordjournals.org/ at Tulane University Library, Serials Acquisitions Dept. on January 13, 2015

incubation. To demonstrate that replicated mitochondrial DNA was distributed in a pattern similar to that of nuclear DNA and to confirm that only parental DNA was used in the repair assays, 50 /tl aliquots of the individual fractions from a typical CsCl gradient were slot blotted and probed for the mitochondrial genome. In Figure 1 positive hybridization was seen in the fractions that corresponded to the parental peak in the CsCl gradient profile from a 0 h culture (fractions 7 — 18). In a 24 h culture, fractions that showed the most intense hybridization signal corresponded to the parental peak (fractions 9-14) and to the replicated peak (fractions 19-30) in the CsCl gradient profile. Similar profiles of hybridization were seen when the blots were stripped and reprobed using a probe for the nuclear sequence containing the insulin gene (data not shown). Densitometric analysis of the slot blots revealed that - 5 0 % of mitochondrial DNA was replicated during the 24 h period evaluated. Scintillation counting of the gradients showed that this value was very similar to thereplicationfound in the overall genome. There was thus no over- or underreplication of the mitochondrial DNA compared to bulk DNA. In all of the repair experiments, only the fractions from the parental peak were pooled and utilized for analysis.

S.P.LeDoux et al.

pyrimidine dimers was detected widiin the mitochondrial genome of CHO cells at either time point. To quantify repair of cisplatin intrastrand adducts, cells were treated with 300 /*M cisplatin for 1 h, and then allowed 0, 8 or 24 h to repair. The DNA was purified, digested with Kpnl and parental DNA was separated. The DNA was then treated with UvrABC endonuclease, separated on a denaturing gel, transferred and probed with the mitochondrial probe. Controls were treated in an identical fashion without UvrABC endonuclease. From the portion of DNA sample that is incised by the endonuclease, the number of incisions per fragment analyzed can be determined.

Previously it has been shown that the UvrABC endonuclease incises about one-half of the cisplatin intrastrand adducts (23). The intensity of the mitochondrial band that reappears at 8 or 24 h (Figure 3) indicates minimal or no repair of cisplatin intrastrand adducts in the CHO mitochondrial genome. Repair of interstrand crosslinks following exposure of CHO cells to 300 /*M cisplatin for 5 h is shown in Figure 4 and in Table I. For these experiments the DNA is alkali denatured, and analyzed on a Southern blot of a neutral gel. DNA that is crosslinked renatures upon neutralization while the rest does not under these low erf conditions and two sharp bands,

TIME

TIME Hrs

24

23.1

Fig. 3. Repair analysis of cisplatin intrastrand crosslinks within the mitochondrial genome of CHO cells. CHO cells were exposed to 300 ^M cisplatin for 1 h and then lysed immediately or allowed to repair for 8 or 24 h. High mol. wt DNA was isolated and digested to completion with Kpnl. Samples were treated (+) or not treated ( - ) with UvrABC endonuclease. The first and second lanes are from 0 h repair cultures. The third and fourth lanes are from 8 h repair cultures. The fifth and sixth lanes are from 24 h repair cultures.

1

2

3

4

5

6

Fig. 5. Repair analysis of alkali-labile sites within the mitochondrial genome of CHO cells following depurination of DNA treated in vivo with nitrogen mustard. CHO cells were exposed to 200 fiM HN2 for 30 min at 37°C in medium containing 1 % serum and then lysed immediately or allowed to repair for 2, 4, 8 or 24 h. High mol. wt DNA was isolated and digested to completion with Kpnl. The first and second lanes are from control cultures that were not heated. The third and fourth lanes are from depurinated (heated) control cultures. The fifth and sixth lanes are from 0 h repair cultures. The seventh and eighth lanes are from 2 h repair cultures. The ninth and tenth are from 4 h repair cultures. The eleventh and twelfth lanes are from 8 h repair cultures. The thirteenth and fourteenth lanes are from 24 h repair cultures. The lower 4.4 kb band is from Hintim pBR322, which was used as an internal loading standard.

TIME Hrs

C

0

8

24 .231

_9.4 _6.6 _4.4 2.3

Fig. 4. Repair analysis of cisplatin interstrand crosslinks within the mitochondrial genome of CHO cells. CHO cells were exposed to 300 jiM cisplatin for 5 h and then lysed immediately or allowed to repair for 8 or 24 h. High mol. wt DNA was isolated and digested to completion with Kpnl. The first and second lanes contain DNA that was not denatured (double-stranded). The third lane contains denatured DNA from cells which were not exposed to cisplatin (single-stranded). The fourth lane contains DNA from 0 h repair cultures. The fifth lane contains DNA from 8 h repair cultures. The sixth lane contains DNA from 24 h repair cultures.

1970

Fig. 6. Repair analysis of alkali-labile sites within the mitochondrial genome of CHO cells following depurination of DNA treated in vivo with MNU. CHO cells were exposed to 500 /iM MNU for 1 h and then lysed immediately or allowed to repair for 8 or 24 h. High mol. wt DNA was isolated and digested to completion with Kpnl. The first and second lanes are from control cultures. The third and fourth lanes are from 0 h cultures. The fifth and sixth lanes are from 8 h repair cultures. The seventh and eighth lanes are from 24 h repair cultures. In these experiments, comparative analysis of the ethidium bromide staining of the gel was used to ensure equal loading.

Downloaded from http://carcin.oxfordjournals.org/ at Tulane University Library, Serials Acquisitions Dept. on January 13, 2015

23.1

ABC ENDO

Repair of mitochondria) DNA

corresponding to single- and double-stranded (crosslinked) DNA, appear. Controls for these experiments consist of lanes containing DNA that was not denatured (double-stranded) in addition to a lane of DNA that was not treated but denatured (single-stranded). As can be seen in Figure 4, after 24 h for repair the crosslinked species (upper band) has nearly completely disappeared, indicating very pronounced repair of this lesion. Exposure of CHO cells to the nitrogen mustard, bis(2-chloroethyl)methylamine (200 /xM for 30 min) resulted in alkylation damage which was not repaired in the mitochondrial genome (Figure 5, Table I) at 8 or 24 h. However, repair was seen in the mitochondrial genome following exposure to the

12

It

2O

Downloaded from http://carcin.oxfordjournals.org/ at Tulane University Library, Serials Acquisitions Dept. on January 13, 2015

t

simple methylating agent MNU (Figure 6). Controls for these experiments consist of DNA from cultures that were not exposed to the drug but were subjected to depurination and alkaline hydrolysis. Based on the break frequencies it was determined that 73% of the alkali-labile sites induced by exposure to MNU were repaired in the mitochondrial genome of CHO cells by 24 h (Table I). In Figure 7, repair of the various lesions in mitochondrial DNA is compared to data previously reported for repair in bulk DNA and repair within the specific nuclear sequence which contains the 5' half of the actively transcribed DHFR gene (17-19,22). In evaluating repair of UV pyrimidine dimers, proficient repair

24

Hrs Repair

Ciiplotin IA

O Bulk OfM • DKFR V UtocH. 4

I

12

II

Hrs Repair

I

12

It

20

24

Hrs Repair

Fig. 7. Comparison of repair efficiency in bulk DNA, DHFR and mitochondrial DNA. The percentage of repair of the various lesions within bulk DNA (O), the nuclear sequence containing the DHFR gene ( • ) , and the mitochondrial DNA sequence (V) is depicted. Repair of pyrimidine dimers and A'-alkylpurines in total genomic DNA was determined by assessing changes in single-stranded mol. wt of the DNA by sedimentation in alkaline sucrose gradients (17-19). The removal of cisplatin lesions in the total genomic DNA was assessed by atomic absorption (22). In the upper left panel, percentage repair of alkali-labile sites following exposure to MNU is compared within the DHFR gene (17) and the mitochondrial DNA sequence. In the upper right panel repair of alkalilabile sites following exposure to nitrogen mustard is compared within bulk DNA, the nuclear DNA sequence containing the DHFR gene (18) and the mitochondrial DNA. The center panel compares the repair of cisplatin intrastrand crosslinks within bulk DNA, the DHFR gene (22) and the mitochondrial DNA sequence. The lower left panel compares the percentage repair of UV dimers within the DHFR gene (19) and the mitochondrial DNA sequence. In the lower right panel, repair of cisplatin interstrand crosslinks within the DHFR gene (22) and the mitochondrial DNA sequence is compared.

1971

S.P.LeDoux tt al.

is seen only in the actively transcribed DHFR gene, while repair across the entire genome and within the mitochondrial DNA is very limited. In comparing repair of cisplatin intrastrand adducts, there was more repair in the actively transcribed DHFR gene than across the entire genome and very limited repair in mitochondrial DNA. Repair of the cisplatin interstrand crosslinks was seen in both the DHFR gene and in mitochondrial DNA. When nitrogen mustard was used as the alkylating agent, repair was detected in the entire genome and in the DHFR sequence but there was no detectable repair in mitochondrial DNA, while repair of A'-methylpurines after exposure of CHO cells to MNU was efficient across the entire genome, in the DHFR gene and in mitochondrial DNA.

1972

The mitochondrial genome is very susceptible to mutation. The mutation rate in mammalian mitochondrial DNA is 5 - 1 0 times greater than in nuclear DNA (38,39). If one compares mutations that occur in a specific nuclear sequence, such as the nuclear encoded subunit of ATP synthase, to mitochondrial genes, mutations occur at a rate that is 17 times faster in the mitochondrial genes (39). These mutations can lead to changes in expression of cell surface antigens (40), decreased respiratory capacity (41) and incorporation of mitochondrial DNA sequences in nuclear DNA (42,43). Based on these observations, it has been hypothesized that these mitochondrial mutations may lead to an acceleration in the release of reactive species of oxygen and that this increase in oxidative stress may contribute to the promotion of cancer or cellular ageing (44). DNA repair processes play a major role in maintaining genetic integrity and therefore protecting against mutations. However, because of the difficulty in isolating sufficient mitochondria] DNA, the study of mitochondrial DNA repair has been an arduous task. Using the methodology developed to study gene-specific DNA repair and a probe that is very specific for the mitochondrial genome, we are now able to evaluate repair of DNA damage in mitochondrial DNA following exposure to a spectrum of DNA-damaging agents. Although there does not appear to be excision repair of UV dimers or bulky alkylation adducts, efficient repair of at least certain lesions such as N-methylpurines is present in mitochondrial DNA. Previously repair of C^-methylguanine (45,46) has also been demonstrated in mitochondrial DNA. Thus, some repair processes are present within the mitochondria. Because of the crucial role that mitochondria play in cellular function, it is imperative that we understand the protective mechanisms associated with these organelles. Acknowledgements We thank Nancy Patton for assistance in the preparation of the photographs for this manuscript. This study was supported in part by NIH Grant ESO3456 and by the Danish Medical Research Council and Danish Cancer Society.

References 1. Anderson.S., Bankler^A.T., Barek.B.G., deBrujin.H.L., Coulson.A.R., Drouin,!., Eperon.I.C, NierUch,P.D., Roe.B.A., Sanger.F., Schreier.P.H.,

Downloaded from http://carcin.oxfordjournals.org/ at Tulane University Library, Serials Acquisitions Dept. on January 13, 2015

Discussion Because of the difficulty in isolating mitochondrial DNA a paucity of information has been available concerning mitochondrial DNA repair. However, utilizing recently developed techniques for assessing sequence-specific DNA repair, we have evaluated the repair of mitochondrial DNA following exposure to a diversified spectrum of DNA damaging agents. Repair of mitochondrial DNA appears to be lesion specific with repair of certain types of lesions and a virtual lack of repair of others. Numerous studies have demonstrated an increased accumulation of damage in mitochondrial DNA (24-32). This increase has been attributed to an increase in initial binding or to a diminished ability to remove the lesion from DNA. From the initial break frequencies, which were determined in the present studies, there appears to be an increase in the alkylation of mitochondrial DNA compared to nuclear DNA when MNU is the damaging agent. This finding is in agreement with previous data where exposure of hamsters and rats to [14C]methylnitrosourea resulted in five times more methylation in mitochondrial DNA (33). With the other agents a large differential in initial damage was not detected; however, the lack of repair that was seen with nitrogen mustard, UV and cisplatin intrastrand adducts would result in an accumulation of damage in mitochondrial DNA. Nuclear DNA repair of UV pyrimidine dimers in CHO cells is very selective with preferential repair of only sequences containing actively transcribed genomic regions (19). In essence, we saw no repair of these dimers in the entire mitochondrial genome. The —10% repair, which was detected, is within the range of noise in the assay and most likely is not repair. Therefore, even actively transcribed areas do not appear to be repaired in mitochondrial DNA. This data is in agreement with that previously reported in HeLa cells (34) and in yeast (35) where a lack of repair of pyrimidine dimers in mitochondrial DNA was also observed. In evaluating DNA repair following exposure to cisplatin, two types of damage were assessed. Repair of the major lesion that is formed following exposure to cisplatin, the intrastrand adduct, was much more efficient in nuclear DNA. By 24 h 42% of the lesions are removed in bulk DNA and 58% are removed in the DHFR gene, compared to 18% in the mitochondrial genome. Thus, it appears that in mitochondrial DNA the rate of removal of cisplatin intrastrand adducts is much slower than in nuclear DNA. Although previous work has indicated that there is no repair of this lesion (36), our more sensitive approach indicates that a low level of repair does indeed take place. In contrast, cisplatin interstrand crosslinks appear to be repaired at similar rates in the mitochondrial genome and in nuclear sequences containing the DHFR gene. While the exact mechanism of this

repair process is unknown, it is thought to be similar to one described in bacteria which involves a recombination event in addition to an excision mechanism (37). In any case, the process appears to be functioning as well in the mitochondria as it is in the nucleus. The more complex alkylation damage produced by nitrogen mustard, which requires nucleotide excision, is not repaired in the mitochondrial DNA CHO cells, while Af-methylpurine damage from exposure to MNU is efficiently removed. These results in combination with the lack of repair of cyclobutane dimers indicate that base excision repair is functional in the mitochondrion and nucleotide excision repair is not. The presence of efficient base excision of N-methylpurines in CHO cells is similar to that previously described for the mitochondrial genome of a rat insulinoma cell line following exposure to the streptozotocin (15). The problem of which enzymes are involved in the repair of mitochondrial DNA must now be addressed. Previously the presence of mitochondrial forms of uracil DNA glycosylase (8-10), AP endonuclease (11), UV-specific endonucleases (12), DNA polymerase (13) and ligase (14) have been demonstrated. Whether these enzymes are involved in repair of Af-methylpurines or whether the nuclear genome has separate genes encoding both nuclear and mitochondrial repair enzymes are questions that remain to be answered.

Repair of mitochondria) DNA

2. 3. 4.

5.

7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26.

27. BackerJ.M. and Weistein.I.B. (1980) Mitochorjdrial DNA is a major cellular target for a dihydrodiol-epoxide derivative of benzo(a)pyrene. Science, 209, 297-299. 28. Niranjan B.G., Bhat.N.K. and Avadhani.N.G. (1982) Preferential attack of mitochondrial DNA by aflatoxin B, during hepatocarcinogenesis. Science, 215, 7 3 - 7 5 . 29. Richter.C, ParkJ.W. and Ames,B.N. (1988) Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. NatL Acad. Sri. USA, 85, 6465-6567. 30. Wunderlich.V., Tetzlaff.I. and Graffi.A. (1972) Studies on nhrosodimethylamine preferential methylan'on of mitochondrial DNA in rats and hamsters. Chem.-BioL Interactions, 4, 81-89. 31. Wilkinson.R., Hawks.A. and Pcgg.A.E. (1975) Methylation of rat liver mitochondrial DNA by chemical carcinogens and associated alterations in physical properties. Chenu-Biol. Interactions, 9, 157 — 167. 32. Miyaki.M., Yatagai.K. and Ono, T. (1977) Strand breaks of mammalian mitochondrial DNA induced by carcinogens. Chenu-Biol. Interactions, 17, 321-329. 33. Wunderlich.V., Schutt.M., Bottger,M. and Graffi.A. (1970) Preferential alkylanon of mitochondrial deoxyribonucleic acid by Mroethyl-A'-nitrosourea. Biochem. J., 118, 99-109. 34. Clayton.D.A., DodaJ.N. and Friedberg.E.C. (1974) The absence of pyrimidine dimer repair mechanism in mammalian mitochondria. Proc. NatL Acad. Sd. USA, 71, 2777-2781. 35. Prakash.L. (1975) Repair of pyrimidine dimers in nuclear and mitochondrial DNA of yeast irradiated with low doses of ultraviolet light. /. Mol. Biol., 98, 781-795. 36. Singh.G. and Maniccia-Bozzo.E. (1990) Evidence for lack of mitochondrial DNA repair following rij-dichlorodiammineplatinum treatment. Cancer Chemother. Pharmacol., 26, 97-100. 37. Zhen.W., Jeppesen.C. and Nielsen.P.E. (1986) Repair of Eschenchia coli of a psoralen-DNA interstrand crosslink site specifically introduced into T410A411 of the plasmid Puc 19. Photochem. Photobiol, 44, 4 7 - 5 1 . 38. Brown,W.M., George.M. and Wilson,A.C. (1979) Rapid evolution of animal mitochondrial DNA. Proc. Natl. Acad. Sd. USA, 76, 1967-1971. 39. Wallace.D.C. (1989) Mitochondrial DNA mutations and neuromuscular disease. Trends Genet., 5, 9—13. 40. Smith.R., Huston.R.N., Jenkin.R.N., Huston.D. and Rich.R.R. (1983) Mitochondria control expression of a murine cell surface antigen. Nature, 306, 599-601. 41. Trumpower,B.L. and Simmons.Z. (1979) Diminished inhibition of mitochondrial electron transfer from succinate to cytochrome by thenoyltrifluroacetone induced by antimycin. /. BioL Chem., 254, 4608-4616. 42. Reid, R.A. (1983) Can migratory mitochondrial DNA activate oncogenes? Trends BioL Sd., 8, 190-191. 43. Richter.C. (1988) Do mitochondrial-DNA fragments promote cancer and aging? FEBS Lett., 241, 1-5. 44. Bandy,B. and Davison.A.J. (1990) Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging? Free Radical Biol. Med., 8, 523-539. 45. Meyers.K.A., Saffhill.R. and O'Conner.P.J. (1988) Repair of alkylated purines in the hepatic DNA in mitochondria and nuclei in the rat. Carcinogenesis, 9, 285-292. 46. Satoh.M.S., Huh.N., Rajewsky.M.F. and Kurolri.T. (1988) Enzymatic removal of O*-ethylguanine from mhcchondrial DNA in rat tissues exposed to N-ethyl-JV-nitrosourea in vivo. J. Biol. Chem.., 263, 6854-6856. Received on May 19, 1992; revised on July 2, 1992; accepted on July 10, 1992

1973

Downloaded from http://carcin.oxfordjournals.org/ at Tulane University Library, Serials Acquisitions Dept. on January 13, 2015

6.

Smith, A.I.H., Staden.R. and Young.I.G. (1981) Sequence and organization of the human mitochondrial genome. Nature, 290, 457-465. Harding.A.E. (1991) Neurological disease and mitochondrial genes. Trends Neurol. Sri., 14, 132-138. Holt.IJ., Harding.A.E. and Morgan-HughesJ.A. (9188) Deletions of muscle mhochondrifll DNA in patients with mitochondrial myopathies. Nature, 331, 717-719. Gerbitz.K.D., Obermaier-Kusser.B., Lestienne.P., Zierz.S., MullerHockerJ., Pongratz,D., Paetzke-BrunnerJ. and Deufer.T. (1990) Mutations of the mitochondrial DNA: the contributions of DNA techniques to the diagnosis of mitochorjdrial encephalomyopathies. J. din. Chem. Oin. Biochem., 28, 241-250. Wallflce.D.C, LotJ.M.T., Lezza.A.M.S., Seibel.P., Volfavec,A.S. and ShoffnerJ.M. (1990) Mitochondrial DNA mutations associated with neuromuscular diseases: analysis and diagnosis using the polymerase chain reaction. Pediatr. Res., 28, 525-528. Rotig,A., Cormier.V., Blancbe.S., BotmefontJ.P., Ledeist,F., Romero.N., SchmitzJ., Rustin.P., Fischer.A., SaudubrayJ.M. and Munnich.A. (1990) Pearson's marrow-pancreas syndrome; a multisystem mitochondrial disorder in infancy. J. Oin. Invest., 86, 1601-1608. Ikebe.S., Tanaka.M., OhnoJC., Sata,W., Hattori.K., Kondo.T., Mizuno.Y. and Ezawa,T. (1990) Increase of deleted mitochondrial DNA in the striatum in Parkinson's disease and senescence. Biochem. Biophys. Res. Common., 170, 1044-1048. Anderson.C.T.M. and Friedberg.E.C. (1980) The presence of nuclear and mitochondrial uracil-DNA glycosylase in extracts of human KB cells. Nucleic Acids Res., 8, 875-888. DomenaJ.D. and Mosbaugh.D.W. (1985) Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver. Biochemistry, 24, 7320-7328. Gupta.P.K. and Sirover.M.A. (1981) Stimulation of the nuclear uracil DNA glycosylase in proliferating humanfibroMasts.Cancer Res., 41, 3133-3136. Tomkinson.A.E., Bonk,R.T. and Iinn.S. (1988) Mitochondrial endonuclease activities specific for apurinic/apyrimidinic sites in DNA from mouse cells. J. Biol. Chem., 263, 12532-12537. Tomkinson,A.E., Bonk.R.T., KimJ., Bartfeld,N. and Linn.S. (1990) Mammalian mitochondrial endonuclease activities specific for ultravioletirradiated DNA. Nucleic Acids Res., 18, 929-935. Bolden.A., Pedrali.N.G. and Weissbach.A. (1977) DNA polymerase is a gamma-polymerase. J. Biol. Chem., 252, 3351-3356. Levin.C.J. and Zimmerman.S.B. (1976) A DNA ligase from mitochondria of rat liver. Biochem Biophys. Res. Common., 69, 514-520. Pettepher.C.C, LeDoux.S.P., Bohr.V.A. and Wilson,G.L. (1991) Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the nitrosourea streptozotocin. J. Bioi Chem., 266, 3113-3117. Kaufman,R.J. and Schimke.R.T. (1981) Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line. Mol. Cell. Biol., 1, 1069-1076. LeDoux.S.P., Thangada.M., Bohr.V.A. and Wilson.G.L. (1991) Heterogeneous repair of methylnitrosourea-induced alkali-labile sites in different DNA sequences. Cancer Res., 51, 775-779. Wassermann.K., Kohn.K.W. and Bohr.V.A. (1990) Heterogeneity of nitrogen mustard-induced DNA damage and repair at the level of the gene in Chinese hamster ovary cells. J. BioL Chem., 265, 13906-13913. Bohr.V.A., Smith.C.A., Okumoto.D. and Hanawalt.P.C. (1985) DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell, 40, 359-369. VosJ.-M.H. and Hanawalt.P.C. (1987) Processing of psoralen adducts in an active human gene: repair and replication of DNA containing monoadducts and interstrand cross-links. Cell, 50, 789-799. Bohr.V.A. and Okumoto.D. (1988) Analysis of pyrimidine dimers in defined genes, p347-366. In Hanawalt,P.C. and Friedburg.E.C. (eds), DNA Repair: A Manual of Research Procedures. Marcel Dekker, New York, Vol. 3. JonesJ.C, Zhen.W., Reed,E., Parker.RJ., Sancar,A. and Bohr.V.A. (1991) Gene specific formation and repair of cisplatin intrastrand adducts and interstrand crosslinks in CHO cells. J. Biol. Chem., 266, 7101-7107. Thomas.D.C, Morton.A.G., Bohr.V.A. and Sancar.A. (1988) General method for quantifying base adducts in specific mammalian genes. Proc. Natl. Acad. Sri. USA, 85, 3723-3733. AUenJ.A. and Coombs.M.M. (1980) Covatent binding of polycydic aromatic compounds to mitochondrial and nuclear DNA. Nature, 287, 244-245. Nagatani.Y. (1960) Cytological studies on the affinity of the carcinogenic azo dyes for cytoplasmic components. Int. Rev. CytoL, 10, 243-313. Takayama.S. and Murumatsu.M. (1969) Incorporation of Undated dimethylnitrosamine into subcellular fractions of mouse liver after long-term administration of dimethylnitrosamine. Z. Krebsfonch., 73, 172-180.

Repair of mitochondrial DNA after various types of DNA damage in Chinese hamster ovary cells.

Using methodology recently developed to assess gene-specific DNA repair, we have demonstrated that it is possible not only to study mitochondrial DNA ...
4MB Sizes 0 Downloads 0 Views