From the Laboratory of Molecular Genetics, NIA, National Institutes of Health, Baltimore, Maryland 21224-6823
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ABSTRACT |
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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)1 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 (1-4) and RNA polymerases (5,
6),2 presumably because TG
induces a local structural change in DNA (7). A negative correlation
has been found between urinary excretion of TG and lifespan of
different mammals (8). In addition, an age-related increase in TG
levels was observed in DNA obtained from rat liver (9). Also, increased
levels of TG have been observed in DNA obtained from various brain
regions from Alzheimer's patients (10). Treatment of cells with the
amyloid 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
(12-17). Additional DNA repair pathways remove TG in mammalians, and
these include nucleotide excision repair (18) and transcription-coupled
repair (19). Transcription-coupled repair of TG was reported to be defective in Cockayne syndrome, a rare autosomal recessive disease with
characteristics of premature aging (19). Repair of TG was found to be
inducible by low doses of ionizing irradiation (20). 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. 21). 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
(22, 23). 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 (24). However, more
recent reports have documented the removal of other types of mtDNA
damage including alkylation lesions (25), cisplatin interstrand
cross-links (25), damage induced by 4-nitroquinoline (26), and
oxidative base damage (27, 28) including endonuclease III-sensitive
sites (29).
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. (30) 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 (31). Pinz and
Bogenhagen (32) recently reconstituted base excision repair of an AP
site with purified Xenopus laevis mitochondrial AP
endonuclease, mtDNA ligase, and mtDNA polymerase 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.
Materials--
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. [ Plasmid Incision Assay--
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 mM
KCl, 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: Oligonucleotide Substrates--
Table
I 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 mM
OsO4 and 2% (v/v)
pyridine.3 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.
For the 5'-end-labeling of the substrates, T4 polynucleotide kinase and
[ Oligonucleotide Incision Assay--
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 mM dithiothreitol, 5 mM EDTA, 0.1 mg/ml bovine serum albumin,
5.5 nM 32P-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 Isolation and Lysis of Rat Liver Mitochondria--
Mitochondria
were purified from rat liver as described (31). 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 µM
phenylmethylsulfonyl 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).
Purification of Mitochondrial Thymine Glycol Recognizing
Enzymes--
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 mM
KCl 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 M
ammonium 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 Rat Liver Mitochondria Contain Enzymatic Activities That Recognize
Osmium Tetroxide-induced DNA Damage--
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 (34,
35). Since OsO4 mainly introduces TG (36), the enzymatic
activity was followed using a radiolabeled TG-containing
oligonucleotide.
Osmium Tetroxide Modification of a Single Thymine-containing
Oligonucleotide Generates One E. coli Endonuclease III-sensitive Site
per 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.
Purification of a Mitochondrial Endonuclease Recognizing Thymine
Glycol--
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.
To exclude that prolonged incubation might induce any additional
oxidative DNA damage in the substrate, we incubated a control oligonucleotide containing a single thymine for up to 18 h with protein extracts obtained at different stages of the purification. No
incision was observed in the thymine-containing control substrate but
only in the TG-containing oligonucleotide (data not shown). Thus, no
potential additional oxidative lesions are formed during the incubation
period that could be substrates for mtTGendo and protein fractions
obtained earlier during the purification.
SDS-polyacrylamide gel electrophoresis of 10 µl of each of the active
Superdex 75 fractions revealed the presence of a major band with a mass
of ~37 kDa upon silver staining (Fig.
4). In inactive fractions 15 and 16, such
a band was not present suggesting that the band we observed represents
mtTGendo. Furthermore, there is a good correlation between the
intensity of the protein signal and the extent of incision (compare the
intensity of the incision product in lanes 17 and
18 (Fig. 4B) with the silver-stained gel bands
(Fig. 4A)) Therefore, by utilizing this purification scheme it seems that we have highly enriched mtTGendo.
mtTGendo Is Associated with the Mitochondrial Inner Membrane or
Matrix--
Fractionation of the purified mitochondria into
submitochondrial fractions was used to demonstrate that mtTGendo was
localized within the mitochondria. Fractionation by digitonin
(according to Ref. 37) produces two submitochondrial fractions, an
outer membrane and a mitoplast fraction. The mitoplast fraction
contains both the inner membrane and matrix components. As shown in
Table III, the outer membrane fraction
was associated with low cytochrome c oxidase activity (a
marker enzyme for the mitoplast fraction) and high monoamine oxidase
activity (a marker enzyme for the outer membrane fraction). Conversely,
the mitoplast fraction was associated with high cytochrome c
oxidase activity and low monoamine oxidase activity. The majority of
the TG incising activity was co-localized with the mitoplast fraction
(Fig. 5 and Table III). This indicates that mtTGendo is associated with the mitochondrial inner membrane or
matrix space. In addition, after incubating the TG-containing oligonucleotide with Pronase-digested mitochondria, incision activity was retained (data not shown), again demonstrating that the observed activity is not of extramitochondrial origin. Thus, we conclude that
mtTGendo is localized within the mitochondria.
Molecular Weight and Catalytic Properties of mtTGendo--
Based
on the calibration of the gel filtration column, mtTGendo eluted at a
position corresponding to a molecular mass range of 25,000-30,000 Da
(Fig. 6). Further fractionation on
Superdex 75 of the two other activities detected after Mono-S
fractionation, from which the peaks eluted at 550 and 650 mM KCl, showed predicted mass of 36 and 42 kDa,
respectively. As shown in Fig. 4, the most pure fractions contained a
single band corresponding to a mass of ~37 kDa, which may represent
mtTGendo. In preliminary experiments, an antibody raised against native
E. coli endonuclease III cross-reacted with the Superdex 75 fractions that contained mtTGendo activity. The antibody cross-reacted
with our putative mtTGendo band.
Further characterization of mtTGendo showed that it is active within a
broad KCl concentration range from 50 to 100 mM. The enzyme
does not require Mg2+ and is resistant to 10 mM
EDTA. Incubation with increasing protein concentrations (0-400 ng of
Fraction V) resulted in increased incision of the TG-containing
oligonucleotide. With 400 ng of protein (Fraction V), maximum incision
(~70% of the oligonucleotide incised) was achieved during 4 h
incubation (data not shown).
Substrate Specificity of mtTGendo--
To address the question of
whether incision by mtTGendo was specific for TG, a set of other
substrates (see Table I) was tested (Fig.
7). Reactions were carried out as
described under "Experimental Procedures." A single-stranded
TG-containing oligo incubated with mtTGendo was not incised
(lane 2). Thus, mtTGendo requires double-stranded
DNA for catalytic activity. To generate an AP site containing oligo
(dsAP), a double-stranded uracil-containing oligonucleotide (dsU) was
first digested with uracil DNA glycosylase. The double-stranded
oligonucleotide containing a single AP site (dsAP) was a substrate for
mtTGendo (lane 7). Partial hydrolysis of this
oligonucleotide to smaller products was observed during the incubation
period (lane 10). However, these products were present in small amounts, and their migration was different than the
main incision product generated by mtTGendo digestion of the AP-containing oligonucleotide. Therefore, mtTGendo has an associated AP
endonuclease activity. A very small amount of incision was observed
when the uracil-containing oligo (dsU) was incubated with mtTGendo
(lane 6). This could be due to incision by the
associated AP endonuclease activity on AP sites generated by
contaminating trace amounts of mitochondrial uracil DNA
glycosylase.
Reaction Products Generated by mtTGendo--
To determine the type
of incision product generated by mtTGendo, the migration of the
incision product was compared with the migration of incision products
generated by some bacterial repair enzymes on a TG or AP site
containing oligo (Fig. 8). EndoIII incises 3' to the lesion through
We then characterized the 5'-end generated by mtTGendo. The thymine
glycol-containing oligo was 3'-labeled with terminal
nucleotidyltransferase and [ Here, we describe the purification and characterization of a
mitochondrial enzyme that specifically excises TG. We name this activity mitochondrial TG endonuclease (mtTGendo) because it recognizes TG and incises the DNA. TG lesions can block mitochondrial DNA and RNA
polymerases, and if not repaired, this can lead to permanent alterations in the mitochondrial genomes. Consequently, mitochondrial function and thereby cellular energy metabolism may be hampered, leading to biological changes associated with aging. Therefore, efficient removal of TG from the mitochondrial genomes is very important.
The activity described here is purely of mitochondrial origin and
is not due to extramitochondrial contamination. We demonstrate that the
majority of the activity was co-localized to mitoplasts (Fig. 5 and
Table III), and after Pronase digestion of the mitochondria, incision
activity specific for the TG was retained.
Tomkinson et al. (33) characterized three mitochondrial
enzymatic activities from mouse plasmacytoma cells, which recognized UV-C-irradiated plasmid DNA. Since mitochondria lack pyrimidine dimer
repair (24), they suggested that the activities recognized TG and other
pyrimidine hydrates. However, no specific substrates were described for
these enzymes. At the UV-C dose employed by these authors (525 J/m2), several other lesions in addition to TG may be
introduced into the DNA, including 8-oxodeoxyguanosine (38-40),
formamidopyrimidine-adenine, and formamidopyrimidine-guanine (41) and
heat-stable dipyrimidine adduct (42). The enzyme activities described
by these authors may be specific for any of these substrates. Thus, it
is unclear whether these activities are similar to the three
mitochondrial TG endonuclease activities that we detect (Fig. 3).
Recently, human (12, 13), Saccharomyces cerevisiae (14, 15),
and Schizosaccharomyces pombe (16) homologs of EndoIII have
been cloned. An amino acid sequence was obtained from a bovine pyrimidine hydrate/thymine glycol DNA glycosylase/AP-lyase (17). In
addition, a predicted amino acid sequence with homology to EndoIII was
obtained from Rattus sp. (17). MtTGendo shares substrate specificity with these eukaryotic enzymes (TG and other pyrimidine hydrates), and all enzymes appear to have an associated AP-lyase function. The molecular mass of mtTGendo (~37 kDa) is comparable to
the reported molecular masses for the EndoIII homologs from higher
eukaryotes (human, 33.6-36 kDa and bovine, 29-31 kDa). Like mtTGendo,
all of the of the eukaryotic homologs are resistant to EDTA. In
addition, all of these enzymes are active within a broad salt
concentration range. The observed mtTGendo activity is not due to
bacterial contamination during the course of isolating the
mitochondria, since the E. coli enzyme is much smaller (23.5 kDa) than mtTGendo (~37 kDa).
Most of the amino acid sequences obtained from eukaryotic homologs of
EndoIII share a conserved iron-sulfur cluster characterized by the
amino acid sequence
Cys-X6-Cys-X2-Cys-X5-Cys
and a helix-hairpin-helix domain, which is thought to be involved in
catalytic activity. Interestingly, S. cerevisiae has two
EndoIII homologs, and one of these, Scr1, encoded by the
FUN33 gene (also called Scr1 (15); NTG1 (14)) lacks the highly conserved iron-sulfur cluster.
Instead, Scr1 was proposed to have a putative mitochondrial
localization signal (15). Scr2 lacks this sequence but has the
iron-sulfur cluster and is thought to be the nuclear counterpart of
Scr1 (15, 43). Although the mass of Scr1 (~45 kDa) is larger than
mtTGendo (~37 kDa), mtTGendo may be a mammalian homolog of Scr1.
Recently, hNTH1 protein, the human homolog of EndoIII (12, 13), was
shown to be localized to both the nucleus and mitochondria (44). This
suggests that the hNTH1 protein may facilitate TG repair in nuclear and
mitochondrial DNA. MtTGendo may be a rat homolog of the
mitochondrial form of hNTH1. Protein microsequencing will be employed
to determine whether mtTGendo shares sequence homology with EndoIII and
its eukaryotic homologs.
Previously, an enzymatic activity from rat liver mitochondria
recognizing 8-oxodG was partially purified and characterized in this
laboratory (31). In contrast to the ability of that enzyme, mtTGendo
does not recognize a double-stranded oligonucleotide containing one
8-oxodeoxyguanosine (dsOG) (Fig. 7, lane 9).
MtODE does not recognize TG-containing
oligonucleotide.4 Also, mtODE
is different from mtTGendo since these enzymes elute at a different KCl
concentration on Mono S column chromatography. Neither of these
mitochondrial base excision repair enzymes require co-factors. In
addition, mtODE has and mtTGendo also seems to have (Fig. 8) an
associated AP-lyase function.
Recently, Pinz and Bogenhagen (32) reconstituted mitochondrial base
excision repair in vitro using an AP site-containing substrate and purified X. laevis mitochondrial proteins. The
AP endonuclease they described is a class II enzyme, cutting 5' to the
abasic site and generating 3'-hydroxyl and 5'-deoxyribosephosphate termini. Both the mtDNA ligase and mtDNA polymerase Mitochondrial DNA exists in a covalently closed circular supercoiled
form. We find that extracts from mitochondria incise an
OsO4-modified supercoiled DNA substrate (Fig. 1),
suggesting that mtTGendo (as well as the two other TG activities)
exists in vivo and acts on TG present in mitochondrial DNA.
As discussed before, processing of AP sites originating from oxidative
damage in mtDNA leads to single-strand breaks. The resulting relaxation of the supercoiled mitochondrial genome may represent a signal for the
recruitment of other DNA repair enzymes or for the initiation of
complete degradation of the oxidized mitochondrial genomes.
We find three separable mitochondrial enzymatic activities for TG (Fig.
3). Although we cannot exclude that these activities may be closely
related forms of the same protein, this finding could implicate that
several mitochondrial mechanisms exist for repair of TG. Therefore,
repair of this lesion from mtDNA could be of critical biological importance.
In conclusion, we have purified a rat liver mitochondrial enzyme
(mitochondrial TG endonuclease) to apparent homogeneity. The enzyme
recognizes TG and shares significant functional homology with other
pyrimidine hydrate-processing base excision repair enzymes. MtTGendo
may help to prevent the accumulation of persistent alterations in
mtDNA. In turn, this may prevent biological alterations associated with
aging and degenerative diseases. Future research will involve the
determination of other substrates for mtTGendo and whether alterations
in the activity of mtTGendo are associated with aging and age-related diseases.
INTRODUCTION
Top
Abstract
Introduction
References
-protein, a protein involved in the pathogenesis of
Alzheimer disease, was found to increase pyrimidine hydrates in
mitochondrial DNA (11). 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.
. Tomkinson
et al. (33) 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.
EXPERIMENTAL PROCEDURES
-32P]ATP
was from NEN Life Science Products and [
-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).
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).
Oligonucleotides used
-32P]ATP were used. Terminal nucleotidyltransferase
and [
-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.
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.
20 °C (Fraction V).
RESULTS
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Fig. 1.
OsO4-treated
PKS+ is nicked upon incubation with fractionated
mitochondrial extract. A, specific incision of
supercoiled (SC) to nicked (N) form of plasmid
damaged with osmium tetroxide. 16.5 µg of DEAE-fractionated
mitochondrial extract or dialysis buffer was added to 50 ng of
untreated or OsO4-treated plasmid and incubated as
described under "Experimental Procedures" for different times at
37 °C. B, sites per plasmid sensitive to digestion by
fractionated extract in untreated and OsO4-treated plasmid.
Open squares, undamaged plasmid; closed circles,
OsO4-treated plasmid. Results represent average
sites/plasmid ± S.D. of duplicate experiments corrected for
background sites/plasmid, with fractionated extract from pooled
mitochondria obtained from two rat livers.
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Fig. 2.
The
OsO4-modified single thymine-containing
oligonucleotide is sensitive to EndoIII but not to EndoIV or Fpg
protein digestion. After OsO4 modification and 5'
32P-labeling, the thymine glycol containing oligo was
digested with EndoIII (lane 6), EndoIV
(lane 7), and Fpg protein (lane 8).
Reaction products were separated on a 20% polyacrylamide, 7 M urea gel. Conversion of the 28-mer substrate to a 10-mer
indicates enzymatic activity. The TG oligo was only incised upon
EndoIII digestion. No incision was observed when the control
thymine-containing oligonucleotide was digested with other bacterial
enzymes (lanes 2-4) or when control and modified
oligonucleotides were incubated with reaction buffer (lanes
1 and 5).
Purification of mtTGendo from rat liver mitochondria
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Fig. 3.
Rat liver mitochondria contain three
enzymatic activities that recognize thymine glycol. A,
4 µl of each Mono S fraction was assayed for incision activity on a
TG-containing oligonucleotide, and the reaction products were separated
on a 20% polyacrylamide, 7 M urea gel. No incision was
observed upon incubation of the Mono S fractions with the
thymine-containing oligonucleotide. B, percent incision of
TG-containing oligonucleotide and KCl concentration of the
corresponding fraction plotted against fraction number.
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Fig. 4.
The Superdex 75 column fractions that contain
mtTGendo activity are highly enriched for a protein with molecular
mass of ~37 kDa. 10 µl of Superdex 75 column fractions were
electrophoresed on a 12% polyacrylamide Tris glycine gel and
silver-stained (A) and 4 µl of each fraction were assayed
for mtTGendo activity (B).
Enzyme activities in fractionated mitochondria
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Fig. 5.
Mitochondrial localization of thymine glycol
recognizing enzymatic activities. Lanes 1 and
2 represent control incubations of reaction buffer with
thymine or thymine glycol oligonucleotide duplexes. Each lane contains
100 µg of protein with T duplex (lanes 3, 5 and
7) or TG duplex (lanes 4, 6, and
8) as indicated under "Experimental Procedures."
Lanes 3 and 4 contain protein from Percoll
gradient purified mitochondria (PG). Lanes 5 and
6 represent the mitoplast protein fraction (MP).
Lanes 7 and 8 were incubated with the
outer membrane fraction (OM). The (small amount of) incision
product produced by the outer membrane fraction has a slightly slower
mobility than the incision products in other lanes due to loading of
the sample in the most outer lane of the gel.
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Fig. 6.
Determination of the molecular weight of
mtTGendo using Superdex 75 column gel filtration. A,
incision activity of each Superdex 75 column fraction was determined as
described under "Experimental Procedures" and plotted against the
elution volumes of proteins used to calibrate the column. B,
extent of incision in each fraction (10-mer incision product is
shown).
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Fig. 7.
MtTGendo recognizes thymine glycol and has an
associated AP-lyase activity. Substrates containing either a
single thymine glycol, apurinic site, uracil, or 8-oxodG were incubated
with 350 ng of mtTGendo for 4 h at 37 °C as described under
"Experimental Procedures." ss T, single-stranded
oligonucleotide containing one single thymine; ss TG,
single-stranded oligo containing thymine glycol; ds T,
double-stranded oligo containing thymine; ds TG,
double-stranded oligo containing thymine glycol; ds APC,
double-stranded AP control oligo; ds AP, double-stranded
AP-containing oligo; ds G, double-stranded G-containing
oligo; ds OG, double-stranded 8-oxodG-containing oligo;
ds U, double-stranded uracil-containing oligo.
-elimination and generates a 3'-unsaturated aldehyde and a 5'-phosphate group. EndoIV incises 5' to
the lesion and generates a 3'-hydroxyl group and a 5'-phosphate deoxyribose residue. Fpg protein incises both 3' (through
-elimination) and 5' (through
-elimination) to the lesion,
releasing the sugar residue and generating 5'- and 3'-phosphate groups.
The chemical structures of the 3' termini created by these different
types of incisions are displayed in Fig. 8B. MtTGendo
produces an incision product on TG or AP-containing oligonucleotides
that migrates like that produced by EndoIII (compare lanes
2 and 3, and lanes 4 and
5). (The additional smaller band seen in Fig. 8, A
and B when both TG and AP oligo were digested with
EndoIII migrates similarly to the incision product generated by Fpg
protein (lane 7), suggesting that part of the
incision product underwent additional
-elimination.)
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Fig. 8.
Determination 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.
-32P[rsqsb]ddATP,
annealed to its complementary oligo, and incubated with mtTGendo or
EndoIII. Both proteins showed a band consistent with a 17-mer
oligonucleotide containing a 5'-phosphate residue (Fig. 8C,
lanes 2 and 6). Together with the incision data
obtained from the 5'-radiolabeled oligo, this also supports that
mtTGendo is an incision activity and not a 3'
5'-exonuclease
stalling at the TG. Thus, like E. coli EndoIII mtTGendo
incises 3' to the lesion and generates a 3'-unsaturated aldehyde and a
5'-phosphate group, suggesting a
-elimination reaction mechanism.
DISCUSSION
were found to
be candidates for the
-elimination reaction required to remove the
remaining 5'-deoxyribose phosphate residue. The resulting one-nucleotide gap was filled in by mtDNA polymerase
and sealed by
mtDNA ligase. If a similar mechanism exists in mammalian mitochondria, the associated AP-lyase function of mtTGendo and mtODE would not be
required in vivo. Mitochondrial class II AP endonucleases
have been isolated from mouse cells (30), suggesting the possibility for the concerted action of mitochondrial DNA glycosylases and AP
endonucleases in mammalian mitochondria. Alternatively, the AP-lyase
function of mtTGendo and mtODE may be required, after which a class II
mitochondrial AP endonuclease, mitochondrial ligase, or polymerase
could process the 3'-unsaturated aldehyde residue further to create a
DNA end suitable for elongation by polymerase
.
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ACKNOWLEDGEMENTS |
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We thank Barbara Hogue for assistance during the purification of mitochondria. We also thank Dr. Richard Cunningham for antibody against E. coli endonuclease III and Dr. Arthur Grollman for Fpg protein. We appreciate advice provided by Drs. Grigory Dianov and Edgar Hudson. We thank Drs. Richard Hansford, Robert Brosh, and R. Michael Anson for critically reading the manuscript. We appreciate the interaction with the Danish Center for Molecular Gerontology.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Molecular and Cellular Biology,
University of California, Berkeley, CA 94720.
§ To whom correspondence should be addressed: Laboratory of Molecular Genetics, NIA, National Institutes of Health, Box 01, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8162; Fax: 410-558-8157; E-mail:vbohr{at}nih.gov.
2 P. Doetsch, personal communication.
3 G. Dianov, unpublished observations.
4 R. Stierum, unpublished observations.
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ABBREVIATIONS |
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The 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.
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REFERENCES |
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