Purification and Characterization of a Mitochondrial Thymine Glycol Endonuclease from Rat Liver
1999; Elsevier BV; Volume: 274; Issue: 11 Linguagem: Inglês
10.1074/jbc.274.11.7128
ISSN1083-351X
AutoresRob Stierum, Deborah L. Croteau, Vilhelm A. Bohr,
Tópico(s)Cancer therapeutics and mechanisms
ResumoMitochondrial DNA is exposed to oxygen radicals produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions in mitochondrial DNA may lead to structural genomic alterations, mitochondrial dysfunction, and associated degenerative diseases. The pyrimidine hydrate thymine glycol, one of many oxidative lesions, can block DNA and RNA polymerases and thereby exert negative biological effects. Mitochondrial DNA repair of this lesion is important to ensure normal mitochondrial DNA metabolism. Here, we report the purification of a novel rat liver mitochondrial thymine glycol endonuclease (mtTGendo). By using a radiolabeled oligonucleotide duplex containing a single thymine glycol lesion, damage-specific incision at the modified thymine was observed upon incubation with mitochondrial protein extracts. After purification using cation exchange, hydrophobic interaction, and size exclusion chromatography, the most pure active fractions contained a single band of ∼37 kDa on a silver-stained gel. MtTGendo is active within a broad KCl concentration range and is EDTA-resistant. Furthermore, mtTGendo has an associated apurinic/apyrimidinic-lyase activity. MtTGendo does not incise 8-oxodeoxyguanosine or uracil-containing duplexes or thymine glycol in single-stranded DNA. Based upon functional similarity, we conclude that mtTGendo may be a rat mitochondrial homolog of the Escherichia coli endonuclease III protein. Mitochondrial DNA is exposed to oxygen radicals produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions in mitochondrial DNA may lead to structural genomic alterations, mitochondrial dysfunction, and associated degenerative diseases. The pyrimidine hydrate thymine glycol, one of many oxidative lesions, can block DNA and RNA polymerases and thereby exert negative biological effects. Mitochondrial DNA repair of this lesion is important to ensure normal mitochondrial DNA metabolism. Here, we report the purification of a novel rat liver mitochondrial thymine glycol endonuclease (mtTGendo). By using a radiolabeled oligonucleotide duplex containing a single thymine glycol lesion, damage-specific incision at the modified thymine was observed upon incubation with mitochondrial protein extracts. After purification using cation exchange, hydrophobic interaction, and size exclusion chromatography, the most pure active fractions contained a single band of ∼37 kDa on a silver-stained gel. MtTGendo is active within a broad KCl concentration range and is EDTA-resistant. Furthermore, mtTGendo has an associated apurinic/apyrimidinic-lyase activity. MtTGendo does not incise 8-oxodeoxyguanosine or uracil-containing duplexes or thymine glycol in single-stranded DNA. Based upon functional similarity, we conclude that mtTGendo may be a rat mitochondrial homolog of the Escherichia coli endonuclease III protein. Reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; mtTGendo, mitochondrial thymine glycol endonuclease; TG, thymine glycol (5,6-dihydroxydihydrothymine); EndoIII, E. coli endonuclease III; AP, apurinic/apyrimidinic site; mt, mitochondria(l); 8-oxodG, 8-oxodeoxyguanosine; mtODE, mitochondrial oxidative damage endonuclease; EndoIV, E. coli endonuclease IV; oligo, oligonucleotide; PKS+, pBluescript II KS+; N, nicked; ss, single-stranded; ds, double-stranded 1The abbreviations used are: ROS, reactive oxygen species; mtTGendo, mitochondrial thymine glycol endonuclease; TG, thymine glycol (5,6-dihydroxydihydrothymine); EndoIII, E. coli endonuclease III; AP, apurinic/apyrimidinic site; mt, mitochondria(l); 8-oxodG, 8-oxodeoxyguanosine; mtODE, mitochondrial oxidative damage endonuclease; EndoIV, E. coli endonuclease IV; oligo, oligonucleotide; PKS+, pBluescript II KS+; N, nicked; ss, single-stranded; ds, double-stranded are generated as by-products of cellular respiration or exogenous exposure to chemical and physical agents. Depending upon the site of formation, ROS can interact with intracellular components including proteins, lipids, and DNA. There is evidence that interactions of ROS with these biological macromolecules play a role in the development of cancer and aging. Upon interaction of ROS with DNA, various adducts can be formed. One of these adducts, the pyrimidine hydrate thymine glycol (TG, 5,6-dihydroxydihydrothymine), is only slightly mutagenic but can block DNA (1Ide H. Kow Y.W. Wallace S.S. Nucleic Acids Res. 1985; 13: 8035-8052Crossref PubMed Scopus (237) Google Scholar, 2Hayes R.C. LeClerc J.E. Nucleic Acids Res. 1986; 14: 1045-1061Crossref PubMed Scopus (90) Google Scholar, 3Clark J.M. Beardsley G.P. Nucleic Acids Res. 1986; 14: 737-749Crossref PubMed Scopus (123) Google Scholar, 4Clark J.M. Beardsley G.P. Biochemistry. 1989; 28: 775-779Crossref PubMed Scopus (52) Google Scholar) and RNA polymerases (5Htun H. Johnston B.H. 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Also, increased levels of TG have been observed in DNA obtained from various brain regions from Alzheimer's patients (10Lyras L. Cairns N.J. Jenner A. Jenner P. Halliwell B. J. Neurochem. 1997; 68: 2061-2069Crossref PubMed Scopus (460) Google Scholar). Treatment of cells with the amyloid β-protein, a protein involved in the pathogenesis of Alzheimer disease, was found to increase pyrimidine hydrates in mitochondrial DNA (11Bozner P. Grishko V. LeDoux S.P. Wilson G.L. Chyan Y.C. Pappolla M.A. J. Neuropathol. Exp. Neurol. 1997; 56: 1356-1362Crossref PubMed Scopus (97) Google Scholar). These studies suggest that due to its possible interference with normal DNA metabolism, the presence of TG in DNA could have biological consequences and might contribute to aging and age-related degenerative diseases. In Escherichia coli, TG is repaired by the base excision repair enzyme endonuclease III (EndoIII). This enzyme is a DNA glycosylase/AP-lyase that first removes the TG and then incises the DNA at the resulting abasic site. Recently, eukaryotic homologs of this base excision repair enzyme have been cloned and characterized (12Hilbert T.P. Chaung W. Boorstein R.J. Cunningham R.P. Teebor G.W. J. Biol. Chem. 1997; 272: 6733-6740Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 13Aspinwall R. Rothwell D.G. Roldan-Arjona T. Anselmino C. Ward C.J. Cheadle J.P. Sampson J.R. Lindahl T. Harris P.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 109-114Crossref PubMed Scopus (255) Google Scholar, 14Eide L. Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar, 15Augeri L. Lee Y.M. Barton A.B. Doetsch P.W. Biochemistry. 1997; 36: 721-729Crossref PubMed Scopus (79) Google Scholar, 16Roldan-Arjona T. Anselmino C. Lindahl T. 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Repair of TG was found to be inducible by low doses of ionizing irradiation (20Le X.C. Xing J.Z. Lee J. Leadon S.A. Weinfeld M. Science. 1998; 280: 1066-1069Crossref PubMed Scopus (186) Google Scholar). The fact that a variety of repair mechanisms exist for TG suggests that repair of this DNA lesion is of critical biological importance. Mitochondrial DNA (mtDNA) consists of a 16.5-kilobase pair circular supercoiled genome that encodes components of the electron transport chain. About 85% of the cellular oxygen consumption is consumed by the mitochondrial electron transport chain (reviewed in Ref. 21Shigenaga M.K. Hagen T.M. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10771-10778Crossref PubMed Scopus (1816) Google Scholar). Since mtDNA is localized in close proximity to the electron transport chain, it is more vulnerable to attack by ROS than nuclear DNA. It is conceivable that the presence of oxidative mtDNA lesions that interfere with mtDNA metabolism leads to mtDNA loss or mutations. Several oxidative DNA lesions have been detected in mtDNA including 8-oxodeoxyguanosine (8-oxodG), 5-hydroxyhydantoin, 5-hydroxymethylhydantoin, 5-hydroxymethylurea, and 5-hydroxycytosine (22Richter C. Park J.W. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6465-6467Crossref PubMed Scopus (1479) Google Scholar, 23Zastawny T.H. Dabrowska M. Jaskolski T. Klimarczyk M. Kulinski L. Koszela A. Szczesniewicz M. Sliwinska M. Witkowski P. Olinski R. Free Radical Biol. & Med. 1998; 24: 722-725Crossref PubMed Scopus (59) Google Scholar). Relatively low levels of EndoIII-sensitive sites have been detected in mtDNA. However, if TG accumulates in mtDNA, mtDNA replication and transcription may be compromised. Consequently, efficient DNA repair of TG may be important for normal mitochondrial function. At present, no study has directly demonstrated the existence of a repair mechanism specific for TG in mitochondria. It has been the notion that mitochondria were devoid of DNA repair since repair of pyrimidine dimers was not observed (24Clayton D.A. Doda J.N. Friedberg E.C. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2777-2781Crossref PubMed Scopus (419) Google Scholar). However, more recent reports have documented the removal of other types of mtDNA damage including alkylation lesions (25LeDoux S.P. Wilson G.L. Beecham E.J. Stevnsner T. Wassermann K. Bohr V.A. Carcinogenesis. 1992; 13: 1967-1973Crossref PubMed Scopus (226) Google Scholar), cisplatin interstrand cross-links (25LeDoux S.P. Wilson G.L. Beecham E.J. Stevnsner T. Wassermann K. Bohr V.A. Carcinogenesis. 1992; 13: 1967-1973Crossref PubMed Scopus (226) Google Scholar), damage induced by 4-nitroquinoline (26Snyderwine E.G. Bohr V.A. Cancer Res. 1992; 52: 4183-4189PubMed Google Scholar), and oxidative base damage (27Taffe B.G. Larminat F. Laval J. Croteau D.L. Anson R.M. Bohr V.A. Mutat. Res. 1996; 364: 183-192Crossref PubMed Scopus (64) Google Scholar, 28Anson R.M. Croteau D.L. Stierum R.H. Filburn C. Parsell R. Bohr V.A. Nucleic Acids Res. 1998; 26: 662-668Crossref PubMed Scopus (68) Google Scholar) including endonuclease III-sensitive sites (29Driggers W.J. LeDoux S.P. Wilson G.L. J. Biol. Chem. 1993; 268: 22042-22045Abstract Full Text PDF PubMed Google Scholar). Although these and other studies suggest the existence of mitochondrial base excision repair, little is known about the mechanism of oxidative DNA damage processing in mitochondria. Purification of mtDNA repair enzymes in sufficient amounts to allow detailed characterization of the repair process is difficult, and eukaryotic oxidative DNA damage processing enzymes are generally expressed at low levels. In addition, the isolation of sufficient amounts of pure mitochondria is a limiting factor in the purification procedure. As a result, only a few mitochondrial DNA repair enzymes involved in base excision repair of oxidative DNA damage have been characterized. Tomkinson et al. (30Tomkinson A.E. Bonk R.T. Linn S. J. Biol. Chem. 1988; 263: 12532-12537Abstract Full Text PDF PubMed Google Scholar) described two class II AP endonuclease-like activities in mitochondria from mouse plasmacytoma cells. In our laboratory, a mitochondrial enzymatic activity specific for 8-oxodG (mtODE) was recently partially purified from rat liver mitochondria (31Croteau D.L. ap Rhys C.M.J. Hudson E.K. Dianov G.L. Hansford R.G. Bohr V.A. J. Biol. Chem. 1997; 272: 27338-27344Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Pinz and Bogenhagen (32Pinz K.G. Bogenhagen D.F. Mol. Cell. Biol. 1998; 18: 1257-1265Crossref PubMed Scopus (164) Google Scholar) recently reconstituted base excision repair of an AP site with purified Xenopus laevis mitochondrial AP endonuclease, mtDNA ligase, and mtDNA polymerase γ. Tomkinsonet al. (33Tomkinson A.E. Bonk R.T. Kim J. Bartfeld N. Linn S. Nucleic Acids Res. 1990; 18: 929-935Crossref PubMed Scopus (72) Google Scholar) identified three mitochondrial endonucleases that recognized DNA lesions induced by high levels of UV light. These enzymes were not well characterized, and no specific substrates were reported. Here, we describe the purification and characterization of a mitochondrial enzyme from rat liver that processes TG and abasic sites. To our knowledge, this is the first direct evidence for the existence of a mitochondrial base excision repair enzyme for TG. The enzyme shares functional similarities with E. coli EndoIII and the mammalian EndoIII homologs. All chemicals were, unless otherwise stated, from Sigma. Livers were obtained from 6-month-old male Wistar rats (Animal Colony of Gerontology Research Center, Baltimore). Percoll and chromatography equipment were from Amersham Pharmacia Biotech. Protease inhibitors were from Boehringer Mannheim. [γ-32P]ATP was from NEN Life Science Products and [α-32P]ddATP was from Amersham Pharmacia Biotech. NensorbTM-20 nucleic acid purification cartridges were from NEN Life Science Products. Osmium tetroxide (OsO4) was purchased from ICN Pharmaceuticals Inc. E. coli endonuclease III (EndoIII) and E. coli endonuclease IV (EndoIV) were from Trevigen. E. coli Fpg protein was kindly provided by Dr. A. Grollmann (Stony Brook, NY). Uracil DNA glycosylase was purchased from Boehringer Mannheim. T4-polynucleotide kinase was from U. S. Biochemical Corp. Terminal nucleotidyltransferase was from Amersham Pharmacia Biotech. Centricon-10 and Centriprep-10 concentrators were obtained from Amicon, Inc. Gel electrophoresis equipment and reagents were from Bio-Rad. pBluescript II KS+ (PKS+) plasmid was obtained from Stratagene. Rabbit polyclonal antibody raised against native EndoIII was a generous gift from Dr. R. Cunningham (Albany, NY). PKS+ plasmid was modified with osmium tetroxide in a reaction mixture (volume, 250 μl) containing 1.2 mm OsO4, 0.4 m NaCl, 50 μg of PKS+ and incubated for 60 min at 70 °C. DNA was precipitated by addition of 0.2 volume of ammonium acetate and 2.5 volumes of 100% ethanol, air-dried, and dissolved in 100 μl of TE, pH 8.0. Supercoiled molecules were isolated from nicked molecules on a sucrose gradient. Plasmid incision assays were performed in the following mixture: 20 mm HEPES, pH 7.6, 75 mmKCl, 5% glycerol, 1 mm EDTA, 0.1 mg/ml bovine serum albumin, and 2 mm dithiothreitol, 50 ng of PKS+, and amounts of protein indicated in the legend of Fig. 1. The reaction mixture was incubated for various times as indicated in Fig. 1. Nicked molecules were separated from supercoiled molecules on a 1% neutral agarose gel and visualized by scanning after ethidium bromide staining using a FluorImagerTM. The intensity of bands representing supercoiled and nicked forms were quantified with ImageQuant software. The number of sites per plasmid were calculated using the formula: −ln(1.4 × intensity of supercoiled form)/(1.4 × intensity of supercoiled form + intensity of nicked form). The presence of OsO4-induced lesions was confirmed by incubating 100 ng of damaged plasmid for 1 h at 37 °C with 0.02–2 units of EndoIII (∼0.8 enzyme-sensitive sites/molecule). TableI shows the sequences of the 28-mer oligonucleotide substrates used in this study. Important features of the oligonucleotide are underlined. The thymine-containing oligonucleotide (T) and its complementary strand were obtained from Life Technologies, Inc. The 8-oxodG (OG), AP site control (APC) oligonucleotide, uracil-containing oligonucleotide (U), its complementary strand, and the complementary strand to the 8-oxodG-containing oligo were from Midland Certified Reagent Co. Oligonucleotides were purified on a 20% polyacrylamide gel prior to use. To generate the thymine glycol (TG)-containing substrate, 1 μg of thymine-containing oligo was incubated for 30 min at room temperature in a 100-μl reaction volume containing 15 mmOsO4 and 2% (v/v) pyridine. 3G. Dianov, unpublished observations. Oligonucleotides were separated from unreacted OsO4 and pyridine using NensorbTM-20 nucleic acid purification cartridges. To generate an AP site containing oligo (AP), the uracil-containing oligo (U) was incubated for 30 min at 37 °C with 1 unit of uracil DNA glycosylase. To demonstrate that AP sites were generated, the AP oligo was incubated for 1 h at 37 °C with 1 unit of EndoIV. About 99% of the AP oligo was incised.Table IOligonucleotides usedNameAbbreviationSequenceThymineT5′ GAA CGA CAG ATG ACA CGA CAG ACA AGC A 3′3′ CTT GCT GTC TAC TGT GCT GTC TGT TCG T 5′Thymine glycolTG5′ GAA CGA CAG ATGG ACA CGA CAG ACA AGC A 3′3′ CTT GCT GTC TAC TGT GCT GTC TGT TCG T 5′UracilU5′ GAA CGA CTG TUA CTT GAC TGC TAC TGA T 3′3′ CTT GCT GAC AAT GAA CTG ACG ATG ACT A 5′AP-site controlAPC5′ GAA CGA CTG TTA CTT GAC TGC TAC TGA T 3′3′ CTT GCT GAC AAT GAA CTG ACG ATG ACT A 5′AP-siteAP5′ GAA CGA CTG TAPA CTT GAC TGC TAC TGA T 3′3′ CTT GCT GAC AAT GAA CTG ACG ATG ACT A 5′GuanineG5′ GAA CGA CTG TGA CTT GAC TGC TAC TGA T 3′3′ CTT GCT GAC ACT GAA CTG ACG ATG ACT A 5′8-Oxo-dGOG5′ GAA CGA CTG TOGA CTT GAC TGC TAC TGA T 3′3′ CTT GCT GAC ACT GAA CTG ACG ATG ACT A 5′ Open table in a new tab For the 5′-end-labeling of the substrates, T4 polynucleotide kinase and [γ-32P]ATP were used. Terminal nucleotidyltransferase and [α-32P]ddATP were used to radiolabel substrates on the 3′-end. NensorbTM-20 nucleic acid purification cartridges were used to separate labeled oligonucleotides from unincorporated nucleoside triphosphates and enzymes. The oligonucleotides were annealed to complementary oligonucleotides by heating to 80 °C in a mixture containing 100 mm KCl, 10 mm Tris, pH 7.8, and 1 mm EDTA and subsequently slowly cooling to room temperature. Incision reactions were performed in a final volume of 20 μl in a mixture containing 20 mm HEPES, pH 7.4, 75 mm KCl, 5 mmdithiothreitol, 5 mm EDTA, 0.1 mg/ml bovine serum albumin, 5.5 nm32P-labeled oligonucleotide duplex, column fractions in the amounts indicated under "Results," and 5–10% glycerol. The reaction was incubated at 37 °C for 4 h-18 h depending on the purity of the fraction tested. Oligonucleotides were precipitated by addition of 4 μl of 11 m ammonium acetate, 1 μl of 20 μg/μl glycogen, and 62 μl of 100% ethanol and pelleted by centrifugation. Pellets were washed with 70% ethanol and dissolved in formamide loading dye consisting of 90% formamide, 0.002% bromphenol blue, and 0.002% xylene cyanol. After heating for 2 min at 80 °C, samples were electrophoresed on a denaturing 20% polyacrylamide, 7 m urea, TBE gel. Because of the heat lability of the AP site, samples containing the AP site oligo were heated to 55 °C prior to loading, instead of 80 °C. Gels were first subjected to autoradiography at −80 °C after electrophoresis. PhosphorScreens were exposed to the frozen gel and quantified using a Molecular Dynamics PhosphorImager combined with ImageQuant software. 1 unit of mtTGendo activity is defined as 1 fmol of TG-containing oligonucleotide incised during a 4-h incubation at 37 °C. Mitochondria were purified from rat liver as described (31Croteau D.L. ap Rhys C.M.J. Hudson E.K. Dianov G.L. Hansford R.G. Bohr V.A. J. Biol. Chem. 1997; 272: 27338-27344Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). All procedures were carried out at 4 °C, unless otherwise indicated. Mitochondria obtained from 4 rat livers (∼8 g) were pooled and resuspended in 15 ml of buffer A with 300 mm KCl. Buffer A consisted of 20 mm HEPES, pH 7.6, 1 mm EDTA, 5% glycerol, 0.015% Triton X-100, 5 mm dithiothreitol. The following protease inhibitors were added prior to use: 1 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml chymostatin A, 2 μg/ml leupeptin, 2 μm benzamide hydrochloride, 1 μmphenylmethylsulfonyl fluoride, and 1 μm E-64. Mitochondria were lysed by slowly adding 10% Triton X-100 to a final concentration of 0.5%. The mitochondrial lysate was subsequently clarified by centrifugation for 1 h at 130,000 ×g in a SW-50.1 rotor (Fraction I). The supernatant obtained after ultracentrifugation was applied to a 25-ml DEAE-Sepharose Fast Flow column, equilibrated with buffer A containing 300 mm KCl. After application of Fraction I to the matrix, the column was washed with 125 ml of the same buffer. 5-ml fractions were collected, and fractions with an absorbance at 280 nm higher than 0.2 absorption units were pooled, and the salt concentration was adjusted to 100 mm KCl (Fraction II). Fraction II was loaded onto a fast protein liquid chromatography HR 10/10 Mono S column (equilibrated with buffer A containing 100 mm KCl). The column was washed with 25 ml of the same buffer and then eluted with a 40-ml linear gradient from 100 mm to 1 m KCl. 1-ml fractions were collected and dialyzed overnight against buffer A containing 100 mmKCl and assayed for mtTGendo activity. Three distinct activities recognizing the TG-containing oligo were detected, and the peaks eluted at ∼410, ∼550, and ∼650 mm KCl, respectively. The most abundant activity eluting at 410 mm KCl was named mtTGendo, and active fractions were pooled, and the buffer was exchanged in a Centricon-10 concentrator for buffer A containing 100 mm KCl without 0.015% Triton X-100. 4 mammonium sulfate (pH 7.6 adjusted with KOH) was slowly added to a final concentration of 1 m (Fraction III), and the sample was applied to a fast protein liquid chromatography HR 5/5 phenyl-Superose column (equilibrated with buffer A containing 100 mm KCl and 1 m ammonium sulfate, without 0.015% Triton X-100). After washing with 5 ml of the same buffer, the column was eluted with a 5-ml linear gradient from 1 to 0 m ammonium sulfate. 400-μl fractions were collected in tubes that contained 0.6 μl of 10% Triton X-100 (to adjust each fraction to a final concentration of 0.015%). Fractions were dialyzed for ∼4 h against buffer A containing 100 mm KCl and then dialyzed against fresh buffer overnight. mtTGendo activity was assayed, and the peak activity was found to elute at ∼230 mm ammonium sulfate. Active fractions were pooled and the buffer was exchanged in a Centricon-10 concentrator for buffer A containing 300 mm KCl and concentrated to a volume of ∼200 μl (Fraction IV). Fraction IV was loaded onto a fast protein liquid chromatography HR 10/30 Superdex 75 gel filtration/size exclusion column equilibrated on buffer A containing 300 mm KCl. The column was calibrated with blue dextran 2000, albumin, ovalbumin, chymotrypsinogen A, and ribonuclease A (low molecular weight standards; Amersham Pharmacia Biotech). The column was eluted with 30 ml of the same buffer, and 400-μl fractions were collected. MtTGendo activity was directly assayed, and active fractions 17 and 18 were pooled and dialyzed against 40 mm HEPES, pH 7.6, 100 mm KCl, 1 mm EDTA, 50% glycerol, 0.015% Triton X-100, 5 mm dithiothreitol, and stored at −20 °C (Fraction V). By using differential centrifugation and Percoll gradient centrifugation purified mitochondria, we identified mitochondrial enzymatic activities that recognize OsO4-induced DNA damage. Initially, we observed nicking of OsO4-modified plasmid upon incubation with a DEAE-fractionated mitochondrial extract. Fig. 1A shows increased conversion of OsO4-modified supercoiled plasmid molecules toward nicked molecules with increasing time of incubation with the DEAE-fractionated extract. In Fig. 1B quantification of the number of sites/plasmids recognized by the DEAE-fractionated mitochondrial extract is shown. A small amount of the observed nicking (∼0.2 sites/plasmid) was nonspecific as observed in the lanes in which unmodified plasmid was incubated with extract. After 24 h of incubation, damage-specific incision was ∼0.4 sites/plasmid (∼50% of the EndoIII-sensitive sites). Incubation of unfractionated extract with plasmid DNA resulted in complete degradation of the substrate, presumably because of mitochondrial endonucleases (34Cummings O.W. King T.C. Holden J.A. Low R.L. J. Biol. Chem. 1987; 262: 2005-2015Abstract Full Text PDF PubMed Google Scholar,35Gerschenson M. Houmiel K.L. Low R.L. Nucleic Acids Res. 1995; 23: 88-97Crossref PubMed Scopus (47) Google Scholar). Since OsO4 mainly introduces TG (36Higgins S.A. Frenkel K. Cummings A. Teebor G.W. Biochemistry. 1987; 26: 1683-1688Crossref PubMed Scopus (52) Google Scholar), the enzymatic activity was followed using a radiolabeled TG-containing oligonucleotide. Upon 32P labeling and digestion of the OsO4-modified thymine-containing oligonucleotide with EndoIII, >80% of the modified oligonucleotide was found to be incised (Fig. 2, lane 6). Migration of the incision product of EndoIII-digested OsO4-modified oligonucleotide was similar to the incision product produced by EndoIII digestion of an oligonucleotide containing a single AP site at the identical position (11th nucleotide relative to the 5′-end of the oligonucleotide, see Fig. 8, A and B, lanes 3 and 5). No other incision products indicative of cytosine modification were observed when the OsO4-modified thymine-containing oligonucleotide was extensively incubated with E. coli EndoIII. This strongly suggests that, in our hands, only the single thymine was converted to TG. No incision was observed upon digestion of the TG oligo with either EndoIV (Fig. 2, lane 7) or Fpg protein (Fig. 2, lane 8) demonstrating that no AP sites or Fpg-sensitive sites were generated during the preparation of the oligonucleotides.Figure 8Determination of the 5′- and 3′-incision products of mtTGendo. A, 5′-labeled oligonucleotide containing either a single thymine glycol or apurinic site was incubated with mtTGendo or with bacterial repair enzymes, and the resulting 10-mers with different 3′-ends were resolved on a 20% polyacrylamide, 7 m urea, TBE gel sequencing gel.B, region of the gel showing 10-mer incision products.C, 3′-labeled oligonucleotides containing a single thymine or thymine glycol were incubated with mtTGendo or with E. coli endonuclease III. For these experiments, mtTGendo was prepared as described except that the phenyl-Superose column chromatography step was omitted. The left panel shows identical migration of the 17-mer incision products generated by digestion of the 3′-labeled TG oligonucleotide with either EndoIII (lane 2) or mtTGendo (lane 6). Lane 7 (right panel) shows migration of the 10-mer incision product generated by EndoIII digestion of 5′-labeled TG oligonucleotide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The purification scheme for the mitochondrial TG endonuclease is summarized in Table II. The mitochondrial lysate (Fraction I) was fractionated on a DEAE-Sepharose Fast Flow column (Fraction II). Fraction II was subjected to fractionation on a Mono S column with a 100–1000 mm KCl linear gradient, and 1-ml fractions were collected. All Mono-S fractions were assayed for incision activity on a TG-containing oligonucleotide. Incision was only observed within Mono-S fractions 18–33. The extent of incision observed in these fractions was quantified and plotted along with the KCl concentration against the fraction number (Fig. 3B). Three separable incision activities that recognize TG were found and the peaks from these activities eluted at ∼410, ∼550, and ∼650 mm KCl. The incision observed was specific for TG, because the oligo does not contain EndoIV or Fpg-sensitive sites (Fig. 2). The most abundant TG-incising fraction eluted at ∼410 mm KCl. The active fractions were pooled, and the KCl concentration was adjusted to 100 mm KCl (Fraction III) and further purified on a phenyl-Superose column (Fraction IV) and a Superdex 75 column (Fraction V). Fraction V was entitled mitochondrial TG endonuclease (mtTGendo). Total and specific activities for each fraction are shown in Table II. We achieved roughly a 450-fold purification.Table IIPurification of mtTGendo from rat liver mitochondriaFractionVolumeProteintotal activityaOne unit of activity is defined as 1 fmol of 28-mer TG-containing oligonucleotide incised per 4-h incubation.specific activityPurificationmlmg103 units103units/mg−foldI. Lysate29.54901370.28II. DEAE185408∼147∼0.36∼1.3III. Mono S0.783.339.61243IV. Phenyl-Superose0.230.0735.575270V. Superdex 750.30.0263.2123440a One unit of activity is defined as
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