Mammalian mitochondrial endonuclease GDigestion of R-loops and localization in intermembrane space Takashi Ohsato1, Naotada Ishihara2, Tsuyoshi Muta1, Shuyo Umeda1, Shogo Ikeda3, Katsuyo
Trang 1Mammalian mitochondrial endonuclease G
Digestion of R-loops and localization in intermembrane space
Takashi Ohsato1, Naotada Ishihara2, Tsuyoshi Muta1, Shuyo Umeda1, Shogo Ikeda3, Katsuyoshi Mihara2, Naotaka Hamasaki1and Dongchon Kang1
1
Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan;2Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan;
3
Department of Biochemistry, Faculty of Science, Okayama University of Science, Japan
Mammalian mitochondria contain strong nuclease activity
Endonuclease G (endoG), which predominantly resides in
mitochondria, accounts for a large part of this nuclease
activity It has been proposed to act as an RNase H-like
nuclease on RNAÆDNA hybrids (R-loops) in the D-loop
region where the origins of mitochondrial replication are
mapped, providing RNA primers for mtDNA replication
However, in contrast with this proposed activity, endoG has
recently been shown to translocate to nuclei on apoptotic
stimulation and act as a nuclease without sequence
specif-icity To clarify the role of endoG in mtDNA replication, we
examined its submitochondrial localization and its ability to
cleave R-loops At low concentration, it preferentially
produces double-stranded breaks in R-loops, but does not act as an RNase H-like nuclease In addition, it exists in the mitochondrial intermembrane space, but not in the matrix where mtDNA replication occurs These results do not support the involvement of endoG in mtDNA replication Based on the fact that guanine tracts, which are preferential targets of endoG, tend to form triplex structures and that endoG produces double-stranded breaks in R-loops, we propose that three-stranded DNA may be the preferred substrate of endoG
Keywords: endonuclease G; mitochondria; mitochondrial DNA; R-loop; triplex DNA
Mammalian mitochondria contain strong nuclease activity
which becomes evident when the membranes are disrupted
by detergents Endonuclease G (endoG) accounts for a large
part of this mitochondrial nuclease activity It is essentially a
nonspecific nuclease for all nucleic acid species including
double-stranded DNA, single-stranded DNA,
single-stran-ded RNA, and RNAÆDNA duplexes [1,2] As endoG
predominantly resides in mitochondria [1], it has been
thought to be involved in the metabolism of mtDNA It is
considered that mitochondrial transcripts stably hybridize
with template strands around conserved sequence blocks
(CSBs) during transcription, forming R-loops consisting of
two DNA strands and one RNA strand, and serve as
primers for mtDNA replication (Fig 1A) EndoG cleaves
the RNA of a linear RNAÆDNA duplex preferentially in the
CSB region [3], raising the possibility that endoG can
generate RNA primers for mtDNA replication [3]
How-ever, endoG is not a specific RNase On the other hand,
RNase MRP
1 , which is also thought to provide RNA
primers by cleaving the RNA of R-loops, is a specific
RNase In addition, the endogenous RNAÆDNA hybrid
is formed in supercoiled mtDNA and should be a
triple-stranded R-loop [4] The cleavage of the RNA of triple-stranded R-loops by endoG has never been shown, while RNase MRP has been shown to cleave the RNA of triple-stranded R-loops preferentially at the CSBs [5] Furthermore, NUC1, which is a yeast homolog of endoG and is also found in mitochondria, is not essential for mtDNA replication in yeast, as disruption of the gene leads
to no obvious derangement of the metabolism of mtDNA [6] Thus the role of endoG in mtDNA replication is still ambiguous
EndoG has recently been reported to be an apoptotic nuclease [7] It translocates to the nucleus upon apoptotic stimulus and extensively degrades nuclear DNA, suggesting that, in mitochondria, it has the potential to fully digest mtDNA This raises another issue of how mtDNA escapes extensive digestion by endoG under steady-state conditions
To further elucidate the function of endoG, we examined its submitochondrial localization and its ability to cleave reconstituted R-loops Here we show that it preferentially produces double-stranded breaks in the R-loops and that it
is localized in the mitochondrial intermembrane space We discuss the possibility that noncanonical DNA structures are preferential substrates for endoG
E X P E R I M E N T A L P R O C E D U R E S
Cloning and expression of human mature endoG
A human cDNA library (Human HeLa S3 MATCHMA-KER cDNA Library; Clontec Laboratories, Palo Alto, CA, USA) was used for amplification of human mature endoG cDNA by PCR A plasmid for mature human endoG with
an N-terminal histidine tag was constructed by inserting the
Correspondence to D Kang, Department of Clinical Chemistry and
Laboratory Medicine, Kyushu University Graduate School
of Medical Sciences, Fukuoka 812-8582, Japan.
Fax: + 81 92 642 5772, Tel.: + 81 92 642 5749,
E-mail: kang@biochem2.med.kyushu-u.ac.jp
Abbreviations: endoG, endonuclease G; CSB, conserved sequence
blocks; mHSP70, mitochondrial heat shock protein 70.
(Received 24 June 2002, revised 2 September 2002,
accepted 6 September 2002)
Trang 2cDNA coding for mature endoG (A49-K297) into
pProEX-HTb(Clontec) and named pHis-hmEG Recombinant
histidine-tagged endoG (His-endoG) was expressed in
Escherichia coli BL21 cells The expressed recombinant
protein formed inclusion bodies The aggregated His-endoG
was denatured and solubilized with buffer containing 6M
guanidine, 0.5MNaCl, 50 mMTris/HCl, pH 8.0, and 1 mM
dithiothreitol The solubilized protein was bound to Ni2+
-chelating Sepharose resin (Amersham Pharmacia Biotech)
The resin was washed with buffer consisting of 20 mMTris/
HCl, pH 7.5, 0.5M NaCl, 5 mM imidazole, 0.1% Triton
X-100, 2 mM 2-mercaptoethanol, 10% glycerol, and 6M
urea The denatured His-endoG was renatured on the resin
by sequentially reducing the urea in the buffer from 6Mto
0M, in 10 steps The renatured His-endoG was finally eluted
with buffer consisting of 20 mM Tris/HCl, pH 7.5, 0.5M
NaCl, 300 mM imidazole, 0.1% Triton X-100, 2 mM
2-mercaptoethanol, and 10% glycerol The eluted sample
was dialyzed against 20 mMTris/HCl, pH 7.5, 0.5MNaCl,
0.1% Triton X-100, 2 mM 2-mercaptoethanol, and 50%
glycerol The recombinant His-endoG was purified to
apparent homogeneity as judged by SDS/PAGE (results not shown) After separation by SDS/PAGE, we deter-mined protein concentrations by Coomassie Brilliant Blue staining using a LAS-1000 CCD camera and IMAGE GAUGETMimage analysis software (Fuji Photo Film) BSA was used as a standard
Cleavage of R-loops by endoG The plasmid pGEMhmD was used for in vitro R-loop formation (Fig 3A) [8] Because we used SP6 RNA polymerase instead of mtRNA polymerase, the human mitochondrial D-loop region lacking authentic promoters for the light and heavy strands was inserted downstream of the SP6 promoter in pGEMhmD R-loops were reconsti-tuted as described previously [8] Briefly, a reaction mixture containing 5 nM pGEMhmD, 50 mM KCl, 20 mM Tris/ HCl, pH 8.0, 10 mMMgCl2, 1 mM dithiothreitol, 0.1 mM NTPs, and 0.2 UÆlL)1SP6 RNA polymerase was incubated for 30 min at 37C essentially as described by Lee & Clayton [9] To remove NTPs, the reaction mixture was applied to a gel-filtration spin column The R-loops in the eluate were ethanol-precipitated, dried, and resolubilzed in distilled water R-loop resolution with RNase H was performed in 20 lL buffer containing 0.1 pmol R-loops,
20 mMTris/HCl, pH 8.0, 50 mMKCl, 4 mMMgCl2, 1 mM dithiothreitol, RNase H (6 U), and 0.05% BSA at 37C for
10 min The reaction was stopped by the addition of
1 lgÆmL)1proteinase K and 0.5% SDS, and then incubated for another 10 min The cleavage reaction was performed in
20 lL buffer containing 0.1 pmol R-loops, 20 mM Tris/ HCl, pH 8.0, 50 mMKCl, 4 mMMgCl2, 1 mM dithiothre-itol, 2 mM ATP, and 0.05% BSA in the presence of the indicated concentration of endoG for 10 min at 37C The reaction was stopped by the addition of 1 lgÆmL)1 prote-inase K and 0.5% SDS, and then incubated for another
10 min R-loops were analyzed by 0.7% agarose gel electrophoresis in buffer consisting of 89 mM Tris base,
89 mMboric acid, and 2 mMEDTA
Determination of cleavage sites After 1 lg R-loops or pGEMhmD had been treated with
50 ng endoG for 10 min, DNA was ethanol-precipitated, washed with 70% ethanol, dried, and solubilized in distilled water To determine the 5¢ ends of cleavage sites, one cycle
of primer extension reactions was performed using 5¢-fluorescein isothiocyanate (FITC)-labeled primers [FD7 (FITC-ctacgttcaatattacaggcg) and FpGEM (FITC-ctttatgcttccggctcgtatg) for the heavy and light strands, respectively] DNA was denatured for 5 min at 95C, the primer was annealed for 0.5 min at 55C, and then an extension reaction was performed for 1 min at 72C using
LA Taq polymerase (Takara, Seta, Japan) For sequence ladders, 25 cycles of primer extension reactions were similarly performed, but using a Thermo Sequence Core Sequencing kit (Amersham Pharmacia Biotech) and 0.5 lg pGEMhmD as a template in the presence of one of the dideoxy dNTPs according to the manufacturer’s instruc-tions The products were resolved on a 7Murea/5% Long RangerTM sequencing gel (FMC Bioproducts, Rockland,
ME, USA) and analyzed with FluorImager 595 (Amersham Pharmacia Biotech)
Fig 1 Cleavage of R-loops (A) The scheme for R-loop formation
in vivo For in vitro R-loop formation, we used SP6 RNA polymerase
instead of mitochondrial RNA polymerase Therefore, in pGEMhmD,
the human mitochondrial D-loop region lacking authentic promoters
for the light and heavy strands is inserted downstream of the SP6
pro-moter (see Fig 3A) LSP, light strand propro-moter; TFAM, mitochondrial
transcription factor A; CSBs, conserved sequence blocks; TAS,
ter-mination associated sequence (B) Supercoiled pGEMhmD plasmids
(upper) or R-loops (lower) were treated with various concentrations of
recombinant human endoG for 10 min at 37 C and then resolved on a
0.7% agarose gel rEG, recombinant human endonuclease G; OC, open
circular form; L, linear form; SC, supercoiled form.
Trang 3Submitochondrial localization of endoG
Anti-endoG sera were obtained by immunizing rabbits with
the recombinant human His-endoG The resulting antisera
also reacted with human, bovine, mouse, and rat endoG
Mouse monoclonal antibodies against cytochrome c and
mitochondrial heat shock protein 70 (mtHSP70) were
purchased from Becton Dickinson and StressGen,
respect-ively Antisera against rat Tim23, Tim43, and Tom20 were
as previously described [10,11]
Rat liver mitochondria were prepared as previously
described [10] For preparation of soluble and particulate
fractions, intact rat mitochondria (1 mg proteinÆmL)1)
were disrupted by sonication in low-salt isotonic buffer
(10 mMHepes/KOH, pH 7.4, 0.22Msucrose, and 0.07M
mannitol), with 0.1MNa2CO3added before sonication in
some experiments Next, 2.0M NaCl and 1.0% Triton
X-100 were added, and the samples were centrifuged at
100 000 g for 30 min and separated into pellets and
supernatants The pellets were suspended in the same
volume of buffer, and each fraction was solubilized with an
equal volume of SDS sample buffer The outer membranes
were disrupted by hypotonic treatment (10 mM Hepes/
KOH, pH 7.4), and the resulting mitoplasts were incubated
with or without proteinase K (100 lgÆmL)1) in the low-salt
isotonic buffer for 1 h at 4C Outer memb ranes were also
disrupted by treatment of intact mitochondria (1 mg
proteinÆmL)1) with digitonin for 10 min in the low-salt
isotonic buffer at 4C Protein concentrations of
mito-chondria were determined by the Lowry method using
BSA as a standard
R E S U L T S
Cleavage of R-loops by endoG
When supercoiled pGEMhmD plasmid was treated with
recombinant human endoG, it was first converted into an
open circular form (Fig 1B, upper panel) With increasing
endoG concentration, a linear form appeared accompanied
by further degraded products It is likely that endoG
introduced nicks into the plasmids, and, in due course, the
accumulation of nicks resulted in double-stranded breaks
We reconstituted R-loops using supercoiled pGEMhmD
plasmids containing the human mitochondrial D-loop
region [8] The R-loops generated showed retarded mobility
on a gel (Fig 2, lane 1 in lower panel) R-loop formation
was confirmed by the restoration of electrophoretic mobility
after treatment with RNase H, which digests the RNA in an
RNAÆDNA duplex (Fig 2, lane 2 in lower panel) The
R-loop mimics the endogenous RNAÆDNA hybrid better
than does a linear RNAÆDNA duplex R-loops were
linearized at concentrations at which endoG converted
ordinary supercoiled plasmids into open circular forms
(Fig 1B, lanes 2 and 3 in upper and lower panels) At higher
concentrations, R-loops were eventually degraded into
small pieces These results suggest that endoG directly
produced double-stranded breaks in the R-loop, instead of
introducing nicks Subsequently, the linearized DNA was
further digested Consistent with this, the linear form
appeared as early as 1 min after the addition of endoG
(Fig 2, lower panel) At this time point, the ordinary
supercoiled plasmid was hardly converted into a relaxed
form at all (Fig 2, upper panel) It is noteworthy that a closed circular form was never observed during endoG treatment of R-loops over the time course of the study (Fig 2 lower panel) If RNA was first cleaved with RNase H activity of endoG, the R-loops would have reverted to closed circular plasmids as they were with RNase H (Fig 2, lane 2 in lower panel) This suggests that cleavage of DNA is a primary event
Fig 2 Time course of endoG digestion Supercoiled pGEMhmD plasmids (upper) or R-loops (lower) were incubated with 50 ng endoG for the indicated periods (lanes 4–10) Supercoiled pGEMhmD plas-mids were incubated without endoG (lanes 2 and 3 in upper panel) R-loops (lane 1 in lower panel) were treated with RNase H (lane 2 in lower panel).
Fig 3 Cleavage sites of R-loops (A) Diagram of pGEMhmD (B) R-loops after cleavage by 50 ng endoG (lane 2), and after further cleavage with ScaI (lane 3) or NaeI (lane 4).
Trang 4We mapped the cleavage sites of the R-loops The
R-loops were treated with endoG and then cleaved with a
restriction enzyme (ScaI or NaeI) that cuts at a unique site
on the plasmid pGEMhmD (Fig 3A) ScaI created two
bands of about 2.1 and 1.9 kbp (Fig 3B, lane 3) NaeI
similarly produced two bands corresponding to about 2.8
and 1.2 kbp (Fig 3B, lane 4) From these results, the
cleavage site by endoG was mapped to the CSB region
(Fig 3A) The positions were further localized by primer
extension The cleavage signals were mapped to the
guanine-rich region in CSB II of the mitochondrial heavy
strand in the ordinary supercoiled plasmids (Fig 4A, lanes
3 and 4) The signals were hardly observed at all at the opposite side, i.e the cytosine-rich region of the light strand (Fig 4B, lane 4), which is consistent with endoG having a preference for a G-tract over an opposite C-tract [12] These observations also explain how endoG produced
an open circular form in the case of ordinary supercoiled plasmids (Figs 1 and 2) In contrast, the cleavage sites in R-loops were clustered between the CSB II and SP6 promoter region in both the heavy (Fig 4A, lanes 7 and 8) and light (Fig 4B, lane 5) strands, and there seemed to be
no sequence specificity We observed nonspecific signals for the heavy strands (Fig 4A, lanes 1–8, asterisk)
Fig 4 Precise mapping of cleavage sites The
2 supercoiled pGEMhmD plasmids and R-loops were incubated with or without EcoRI or endoG (A) Heavy strand The cleavage sites were determined by primer extension using the primer FD7 Lanes T, G, C, and A indicate sequence ladders (lanes 9–12) Except for the sequence lanes, each sample was duplicated; the two lanes correspond to 0.5 lg (lanes 1, 3, 5, and 7) and 1.0 lg (lanes 2, 4, 6, and 8) of template DNA, respectively Signals marked with * may be nonspecific artifacts caused by stalling of the extension reaction (B) Light strand The reactions were performed as in (A), except the primer FpGEM was used Except for the sequence lanes (lanes 6–9), each sample corresponds to 1.0 lg template DNA (lanes 1–5).
Trang 5Because these signals were observed even in nondigested
plasmids (Fig 4A, lanes 1 and 2), the DNA polymerase
used in the primer extension reaction may tend to pause
around the sites, and the signals may be technical artifacts
We also saw several signals for the light strand when
nondigested R-loops were used (Fig 4B, lane 3) Because
such signals were not observed when normal pGEMhmD
was used (Fig 4B, lane 2), primer extension reactions
would frequently and artificially terminate only when using
R-loops Although the exact reason for the termination is
not clear, considering that R-loops are fairly stable to heat
[9] and the template light strand was originally hybridized
with RNA, one possibility is that RNAÆDNA hybrids that
remain even after denaturation by heat may block the
extension reaction
Submitochondrial localization of endoG
Next we examined the submitochondrial localization of
endoG When rat liver mitochondria were disrupted by
sonication in low-salt isotonic buffer, endoG was mainly
recovered from a particulate fraction (Fig 5A, lanes 7 and
8) On the other hand, it was released into the soluble
fraction in the presence of 2.0MNaCl, 0.1MNa2CO3, or
0.5% nonionic detergent Triton X-100 (Fig 5A, lanes
1–6), indicating that it is peripherally associated with the
mitochondrial membranes Cytochrome c, a protein
loosely associated with inner membranes on the
inter-membrane-space side, showed the same behavior as
endoG (results not shown) EndoG in intact mitochondria
was resistant to proteinase K (Fig 5B, lanes 1 and 2), but
when the outer membranes were disrupted by hypotonic treatment, it was digested by proteinase K (Fig 5B, lanes
3 and 4) This indicates that it is localized in the intermembrane space, because the inner membranes remained intact, protecting mHSP70 (a mitochondrial matrix protein) from digestion (Fig 5B, lane 4) Although
a trace of endoG remained even after the proteinase K treatment (Fig 5B, lane 4), cytochrome c also remained to
a similar extent (Fig 5B, lane 4), suggesting that the residual endoG is due to incomplete disruption of the outer membranes To confirm this localization, the outer membranes were disrupted with digitonin and then digested with proteinase K At 500 lgÆmL)1 digitonin (Fig 5C, lane 3), Tim23, an inner membrane protein facing the intermembrane space, was completely digested with proteinase K, whereas neither mHSP70 nor Tim44,
an inner membrane protein facing the matrix side, was digested This indicates that the protease has access to the outside of the inner membranes but not the matrix side Under these conditions, both cytochrome c and endoG were digested (Fig 5C, lane 3) Both mHSP70 and Tim44 were completely digested with proteinase K when mito-chondria were solubilized with 0.5% Triton X-100 (results not shown) Thus, endoG and mtDNA are localized to different compartments, which may protect mtDNA from extensive digestion by endoG
D I S C U S S I O N
EndoG is essentially a nonspecific nuclease [1,2], which is evident in vivo after apoptotic stimulation, when endoG
Fig 5 Submitochondrial localization of endoG (A) Intact rat mitochondria (lane 9) were disrupted by sonication in low-salt isotonic buffer (lanes 7 and 8) Then 2.0 M NaCl (lanes 5 and 6) and 1.0% Triton X-100 (lanes 1 and 2) were added In some preparations, 0.1 M Na 2 CO 3 was added before sonication and there was no further treatment (lanes 3 and 4) The samples were centrifuged and separated into pellets (P) and supernatants (S) Samples corresponding to 20 lg mitochondrial protein were applied to each lane EndoG was detected by Western blotting (B) Intact mito-chondria were incubated without proteinase K (lane 1) or with proteinase K (lane 2) for 1 h at 4 C Outer membranes were disrupted by hypotonic treatment The mitoplasts were then incubated without proteinase K (lane 3) or with proteinase K (lane 4) in the low-salt isotonic buffer for 1 h at
4 C Tom20, endoG, cytochrome c, Tim44, and mHSP70 were detected by Western blotting (C) Intact mitochondria were treated with digitonin for 10 min in the low-salt isotonic buffer at 4 C followed by proteinase K digestion (lanes 1–3) mHSP70, Tim44, Tim23, endoG, and cytochrome c were detected by Western blotting.
Trang 6translocates en masse from mitochondria to nuclei and
extensively digests nuclear DNA [7] Under these
condi-tions, endoG at high concentration appears to act as a
nonspecific nuclease However, at low concentration, it
appears to be quite specific for kinked DNA, such as that in
R-loops (Fig 1) We have shown that endoG preferentially
introduced double-stranded breaks in a specific region of
R-loops
RNA consistently forms a hybrid in CSB II [9], and
transcription for R-loop formation starts from the SP6
region in our in vitro transcription system Therefore, the
starting point of the RNAÆDNA hybrid formation must
be located between these two regions Considering that
there was no sequence specificity, endoG may recognize a
three-stranded junction structure formed at the starting
point of the RNAÆDNA hybrid Alternatively, it may
recognize the looping-out single-stranded DNA and an
A-form helix that an RNAÆDNA duplex is likely to take
on As reported elsewhere, endoG preferentially cleaves
damaged DNA [13] Many kinds of DNA damage cause
distortion of the DNA helix Guanine-rich DNA, which
is a preferential target of endoG [12], tends to form
irregular structures such as triplexes and quadruplexes
[14] Taken together with our observations of the activity
of endoG on R-loops, it is evident that noncanonical
structures of DNA, e.g damaged DNA, triplex DNA,
and R-loops, may generally be preferential substrates for
endoG
Triplex or quadruplex DNA strands can be formed
intramolecularly or intermolecularly in vivo in purine-rich
regions [14] These structures block transcription and
replication For instance, it has been reported that
expansion of the GAA triplet repeat of the frataxin
gene results in triplex structures, thereby causing
tran-scription to be paused [15] R-loop formation can occur
in a nonspecific manner during transcription [16] and can
serve as a primer for DNA synthesis [17], although the
nascent RNA molecule is normally displaced from the
DNA template strand shortly after synthesis
Accumula-tion of R-loops is hazardous to cells, because of
induction of unregulated replication [18] In addition,
guanine-rich RNA can participate in very stable
three-stranded structures by establishing both Watson–Crick
and Hoogsteen-type hydrogen bonds [19], the formation
of which inhibits transcription Given that endoG is
normally regulated to exist in nuclei at a very low level,
it may have a role in survival in normal states
by eliminating kinked DNA formed in guanine-rich
regions
EndoG has been proposed to provide primers for
mtDNA replication by cleaving the RNA moiety of
RNAÆDNA hybrids formed at the origin of replication [3]
However, based on our observations, endoG would not
act as an RNase in these conditions, but would instead
preferentially degrade mtDNA in an R-loop
Further-more, we have shown that endoG exists in the
intermem-brane space (Fig 5B,C), whereas mtDNA replication
should take place in the matrix On the other hand,
RNase MRP has been shown to cleave RNA of
triple-stranded R-loops preferentially at the CSBs [5], and is
another RNase proposed to create RNA primers The role
of endoG in mtDNA replication may require careful
re-evaluation
A C K N O W L E D G E M E N T S
This work was supported in part by Grants-in Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports, and Culture of Japan.
R E F E R E N C E S
1 Gerschenson, M., Houmiel, K.L & Low, R.L (1995) Endonuclease G from mammalian nuclei is identical to the major endonuclease of mitochondria Nucleic Acids Res 23, 88–97.
2 Widlak, P., Li, L.Y., Wang, X & Garrard, W.T (2001) Action of recombinant human apoptotic endonuclease G on naked DNA and chromatin substrates: cooperation with exonuclease and DNase I J Biol Chem 276, 48404–48409.
3 Cote, J & Ruiz-Carrillo, A (1993) Primers for mitochondrial DNA replication generated by endonuclease G Science 261, 765–769.
4 Shadel, G.S & Clayton, D.A (1997) Mitochondrial DNA main-tenance in vertebrates Annu Rev Biochem 66, 409–435.
5 Lee, D.Y & Clayton, D.A (1997) RNase mitochondrial RNA processing correctly cleaves a novel R loop at the mitochondrial DNA leading-strand origin of replication Genes Dev 11, 582–592.
6 Zassenhaus, H.P., Hofmann, T.J., Uthayashanker, R., Vincent, R.D & Zona, M (1988) Construction of a yeast mutant lacking the mitochondrial nuclease Nucleic Acids Res 16, 3282–3296.
7 Li, L.Y., Luo, X & Wang, X (2001) Endonuclease G is an apoptotic DNase when released from mitochondria Nature (London) 412, 95–99.
8 Ohsato, T., Muta, T., Fukuoh, A., Shinagawa, H., Hamasaki, H.
& Kang, D (1999) R-loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction-specific helicase Biochem Biophys Res Commun 255, 1–5.
9 Lee, D.Y & Clayton, D.A (1996) Properties of primer RNA– DNA hybrid at the mouse mitochondrial DNA leading origin of replication J Biol Chem 271, 24262–24269.
10 Ishihara, N & Mihara, K (1998) Identification of the protein import components of the rat mitochondrial inner membrane, rTIM17, rTIM23, and rTIM44 J Biochem (Tokyo) 123, 722– 732.
11 Iwahashi, J., Yamazaki, S., Komiya, T., Nomura, N., Nishikawa, S., Endo, T & Mihara, K (1997) Analysis of the functional domain of the rat liver mitochondrial import receptor Tom20.
J Biol Chem 272, 18467–18472.
12 Ruiz-Carrillo, A & Renaud, J (1987) Endonuclease G: a (dG) n X (dC) n-specific DNase from higher eukaryotes EMBO J 6, 401–407.
13 Ikeda, S & Ozaki, K (1997) Action of mitochondrial endonuclease G on DNA damaged by L -ascorbic acid, peplomy-cin, and cis-diamminedichloroplatinum (II) Biochem Biophys Res Commun 235, 291–294.
14 Gilbert, D.E & Feigon, J (1999) Multistranded DNA structures CurR Opinion Sruct Biol 9, 305–314.
15 LeProust, E.M., Pearson, C.E., Sinden, R.R & Gao, X (2000) Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich’s ataxia J Mol Biol 302, 1063–1080.
16 Masse, E & Drolet, M (1999) Escherichia coli DNA topoisome-rase I inhibits R-loop formation by relaxing transcription-induced negative supercoiling J Biol Chem 274, 16659–16664.
17 Asai, T & Kogoma, T (1994) D-loops and R-loops: alternative mechanisms for the initiation of chromosome replication in Escherichia coli J Bacteriol 176, 1807–1812.
18 Hong, X., Cadwell, G.W & Kogoma, T (1995) Escherichia coli RecG and RecA proteins in R-loop formation EMBO J 14, 2385–2392.
19 Praseuth, D., Guieysse, A.L & Helene, C (1999) Triple helix formation and the antigene strategy for sequence-specific control
of gene expression Biochim Biophys Acta 1489, 181–206.