Mitochondrion 5 (2005) 3544

www.elsevier.com/locate/mito

DNA-binding proteins of mammalian mitochondria

Michael P. Kutsyi*, Natalia A. Gouliaeva, Elena A. Kuznetsova, Azhub I. Gaziev
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290, Russia
Received 15 June 2004; received in revised form 1 September 2004; accepted 9 September 2004

Abstract
Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was
investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular
mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNAprotein complexes at a physiological NaCl
concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between
DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and
nDNA. In the presence of 5 mg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected
little the binding of proteins to DNA. There was no distinct difference in DNAprotein complex formation of liver
mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble
proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are
activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the
regulation of the structural organization and functional activity of mitochondrial DNA.
q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Keywords: Mitochondria; DNA-binding proteins; Acid-soluble proteins; Histones; Proteases

1. Introduction
In mammalian somatic cells, the mitochondrial
DNA (mtDNA) is represented by numerous copies
(from 1 to several thousands) of double-stranded
circular molecules of 16.5 kb (Anderson et al., 1981;
Bibb et al., 1981; Takamatsu et al., 2002). DNA in
mitochondria is mostly associated with the inner
membrane (Adams et al., 1980; Potter et al., 1980).
The mtDNA interacts with proteins of the inner

* Corresponding author. Fax: C7 967 330553.

E-mail address: m_kutsyi@rambler.ru (M.P. Kutsyi).

mitochondrial membrane via particular D-loop

regions (Potter et al., 1980). MtDNA compared to
nuclear DNA (nDNA) is more vulnerable to reactive
oxygen species (ROS) formed in the mitochondrial
respiratory chain. The number of oxidized bases in
mtDNA is about 16 times as high as in nDNA (Richter
et al., 1988; Richter, 1992). Mutations and deletions in
mtDNA may be a cause of aging and development of
degenerative diseases in the organism (Wallace, 1992;
Cortopassi et al., 1992). It has been suggested that a
major contributor to the high sensitivity of mtDNA to
damaging ROS influences is the absence of histones in
mitochondria. Therefore, in intact mitochondria DNA
is not covered by histones and other proteins, as it is

1567-7249/$ - see front matter q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
doi:10.1016/j.mito.2004.09.002

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M.P. Kutsyi et al. / Mitochondrion 5 (2005) 3544

the case in the nuclei (Richter, 1992; Wallace, 1992;

Higuchi and Linn, 1995). At the same time, some
authors have shown the presence of mtDNAprotein
complexes, in which protein is tightly associated with
DNA and this linkage is stable to the action of ionic
detergents and high concentrations of NaCl. In
particular, DNAprotein complexes have been isolated from rat liver mitochondria after their lysis with
sarkosyl (Van Tuyle and Kalf, 1972) or with BRIJ 58
in the presence of sodium deoxycholate and 0.5 M
NaCl (Van Tuyle and McPherson, 1979). According
to electron microscopy data, mtDNA molecules are
organized as rosette-like structures with a dense
central core (Van Tuyle and McPherson, 1979).
They are maintained in the highly folded state by
associated proteins. DNAprotein complexes have
also been isolated from the yeast S. cerevisiae
mitochondria (Rickwood et al., 1981; Miyakawa
et al., 1987). From liver mitochondria, a histone-like
protein of 16 kDa molecular mass has been isolated
which interacts with nucleotide sequences in the
region of the D-loop of mtDNA (Pavco and Van
Tuyle, 1985). Histone-like proteins have also been
obtained from the mitochondria of heart (Yamada
et al., 1991), yeast (Caron et al., 1979), and Xenopus
laevis (Mignotte et al., 1985). Recently it has been
shown that multiple mtDNA molecules are organized
into discrete DNAprotein complexes called
nucleoids (Miyakawa et al., 1987; Garrido et al.,
2003). At the present time, the protein composition of
mitochonrial nucleoids and the mechanisms by which
mtDNA functions within this dynamic DNAprotein
structure have not yet been completely elucidated. It
has been shown that nucleoids from the mitochondria
of primary human fibroblast cells, 143B osteosarcoma
and A549 adenocarcinoma cells contain mitochondrial gamma-polymerase, the transcriptional factor A
of mitochondria, and mitochondrial single-stranded
DNA binding proteins (Garrido et al., 2003). Probably, proteins loosely bound to DNA also participate
in the packing of mtDNA within the nucleoid. When
DNA or DNAprotein complexes are isolated under
rigorous conditions with the use of ionic detergents or
high salt concentrations, the proteins loosely bound to
DNA dissociate and hence are not detected in isolated
preparations of DNA and DNAprotein complexes.
Recently, proteins binding to RNA have been shown

to be present in mammalian mitochondria (Koc and

Spremulli, 2003).
In the present work, a spectrum of acid-soluble
DNA-binding proteins of rat liver and spleen
mitochondria is presented and their ability to form
complexes with DNA is shown. Also it is shown that
these acid-soluble proteins contain proteases including DNA-activated ones.

2. Materials and methods

2.1. Materials
All chemical reagents were of analytical grade. All
reagents for polyacrylamide gel electrophoresis
(PAGE), calf thymus DNA, DNaase, RNaase A,
antipain, leupeptin, phenylmethylsulphonyl fluoride
(PMSF), N-ethymaleimide (NEM), and digitonin
were obtained from Sigma Chemical Company.
Proteinase K and sodium dodecyl sulfate (SDS)
were obtained from AppliChem GmbH (Germany),
lambda DNA from phage isolated from the E. coli
W3110 (c/1857 Sam7) strain, protein molecular mass
standards (b-galactosidase, 116.0 kDa; bovine serum
albumin, 66.2 kDa; ovalbumin, 45.0 kDa; lactate
dehydrogenase, 35.0 kDa; restriction endonuclease
Bsp981, 25.0 kDa; b-lactoglobulin, 18.4 kDa; lysozyme, 14.4 kDa) were obtained from Fermentas MBI.
2.2. Isolation of nuclei and mitochondria
Male Wistar rats weighing 180200 g were used.
Rat liver and spleen mitochondria were isolated by
differential centrifugation, as described by Pedersen
et al. (1978). Mitochondria were purified from
lysosomal impurities by treatment with digitonin
(Loewenstein et al., 1970). Nuclei from liver and
spleen were isolated as described by Blobel and Potter
(1966). DNA from liver mitochondria was isolated
according to Suter and Richter (1999).
2.3. Extraction of acid-soluble proteins from nuclei
and mitochondria
Acid-soluble proteins (histones) were extracted
from liver and spleen nuclei with 0.2 M H2SO4, as
described by Bonner et al. (1968). Histones thus

M.P. Kutsyi et al. / Mitochondrion 5 (2005) 3544

obtained were precipitated with ethanol and extracted

once again with 0.2 M H2SO4. Analogously, acidsoluble proteins were extracted from liver and spleen
mitochondria. Protein concentration was determined
according to Lowry et al. (1951).
2.4. Electrophoresis of proteins in polyacrylamide gel
Electrophoresis of acid-soluble proteins was performed in 15% SDS-polyacrylamide gel by the
method of Laemmly (1970). Enzyme-electrophoresis
of proteases associated with acid-soluble proteins of
mitochondria was carried out in 15% SDS-polyacrylamide gel containing covalently bound casein according to Kelleher and Juliano (1984) with minor
modification. Acid-soluble mitochondrial proteins
were dissolved in a buffer containing SDS but with
no 2-mercaptoethanol. Samples were not boiled
before being applied to gel. After electrophoresis,
the gel was washed for 1.5 h with 0.02 M TrisHCl
buffer, pH 8.0 containing 0.5% Triton X-100 to
remove SDS. In order to detect protease activity, the
gel was incubated for 48 h at 37 8C in 0.02 M Tris
HCl buffer, pH 8.0, containing 25 mM NaCl. After
incubation, the gel was stained with Coomassie
brilliant blue (CBB R-250).
2.5. Formation of DNAprotein complexes
To obtain DNAprotein complexes, different
amounts of DNA (2, 5, and 10 mg) were added to
50 mg of acid-soluble mitochondrial proteins in
0.02 M TrisHCl, pH 7.4, containing 0.15 M NaCl.
In order to study the effect of NaCl on the formation
of DNAprotein complexes, 0.150.5 M NaCl was
added to 50 mg of protein and 10 mg of DNA. Thirty
minutes later, DNAprotein complexes were sedimented by centrifugation (5000!g, 30 min at 4 8C).
Then the pellet and supernatant were subjected to
electrophoresis in 15% SDS-polyacrylamide gel.
The formation of DNAprotein complexes was
also judged from comparing the binding of actinomycin D to free DNA and DNA complexed with
protein (Muller and Crothers, 1968). In the case
of DNAprotein complex formation, the binding of
actinomycin D must be lower than in the case of
free DNA.

37

2.6. Assay of protease activity

In order to determine the activity of proteases
associated with acid-soluble mitochondrial proteins,
50 mg of proteins in 0.04 M TrisHCl buffer, pH 8.0,
were incubated for 1.53.0 h at 37 8C in the presence
or in the absence of 10 mg of native or denatured
DNA. DNA was denatured by heating for 10 min at
100 8C and then rapidly cooled. After incubation,
samples were subjected to electrophoresis in 15%
SDS-polyacrylamide gel. After staining with Coomassie brilliant blue, gels were scanned with
subsequent computation. Protease activity was
judged from the amount of polypeptides degraded
in the course of incubation. In estimating the pH
dependence of the activity of proteases associated
with mitochondrial acid-soluble proteins, these
proteins were incubated for 3 h at 37 8C and the
amount of degraded proteins was determined by the
method of Lowry et al. (1951).

3. Results
3.1. DNA-binding acid-soluble proteins
of mitochondria
Acid-soluble proteins were isolated from mitochondria and nuclei of rat liver and spleen by
extraction with 0.2 M H2SO4. It should be noted that
extraction with 0.2 M H2SO4 is commonly used to
isolate basic proteins, histones, from the nuclei
(Bonner et al., 1968). As is seen from electrophoregrams in Fig. 1 (lanes 3, 5), the basic proteins from
liver and spleen nuclei are represented by five histone
fractions, which is consistent with the literature data
(Bonner et al., 1968; Hnilica, 1975; Olszewska and
Tait, 1980). However, in our experiments the treatment
of liver and spleen mitochondria with 0.2 M H2SO4
also gave acid-soluble proteins. Upon electrophoresis
in 15% SDS-polyacrylamide gel, these proteins were
divided into more than 20 polypeptides with molecular
masses of 10120 kDa. The electrophoretical spectra
of acid-soluble proteins from spleen mitochondria are
similar to those from liver mitochondria, although
there are distinct differences in the ratios between
particular peptides (Fig. 1, lanes 2, 4). As upon the
treatment of nuclei with sulfuric acid the basic

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M.P. Kutsyi et al. / Mitochondrion 5 (2005) 3544

Fig. 1. Acid-soluble proteins of mitochondria and nuclei from rat

liver and spleen. Lane 1, protein markers; Lane 2, liver
mitochondrial proteins; Lane 3, liver nuclear proteins (histones);
Lane 4, spleen mitochondrial proteins; Lane 5, spleen nuclear
proteins (histones).

proteins, histones, are extracted, it can be assumed that

the acid-soluble proteins extracted by the same
procedure from mitochondria may mostly possess the
properties of basic proteins.
3.2. Formation of nucleoproteid complexes:
acid-soluble proteinsDNA
In further experiments, it was found that acidsoluble proteins from liver and spleen mitochondria
form nucleoproteid complexes with DNA at the
physiological NaCl concentration. These complexes
are easily isolated by centrifugation from the DNA
and proteins that are unable to form complexes and
remain in the supernatant (Fig. 2). There is no
significant difference between DNA-binding proteins

Fig. 2. Formation of mitochondrial acid-soluble proteinsDNA

complexes. MtDNA (10 mg) or nDNA (10 mg) were added to 50 mg
of spleen mitochondrial proteins in 0.02 M TrisHCl buffer, pH 7.4,
containing 0.15 M NaCl. After centrifugation, the proteins of pellet
(P) and those of supernatant (S) were subjected to electrophoresis.
Lane 1; initial proteins (50 mg). Lanes 2 and 3; protein and mtDNA.
Lanes 4 and 5; protein and nDNA.

of mitochondria from liver and those from spleen as to

their ability to form complexes with mtDNA and
nDNA. In studying the dependence of DNAprotein
complex formation on the amount of nDNA or
mtDNA, it was found that addition of 2 mg of DNA
to the reaction mixture resulted in the binding of
substantial amounts of mitochondrial acid-soluble
proteins (Fig. 3). In the presence of 5 mg of DNA most
polypeptides were bound to DNA, and further
increase of DNA amount to 10 mg influenced little
the binding of proteins to DNA. There was no distinct
difference in DNAprotein complex formation of
liver mitochondrial acid soluble proteins with
nDNA or mtDNA (Fig. 3). The study of the NaCl
concentration dependence of the binding of liver

M.P. Kutsyi et al. / Mitochondrion 5 (2005) 3544

39

Fig. 3. Dependence of acid-soluble proteinDNA complex formation on DNA concentration. MtDNA or nDNA (2, 5, 10 mg) were added to
50 mg of liver mitochondrial proteins in 0.02 M TrisHCl buffer, pH 7.4, containing 0.15 M NaCl. After centrifugation, the proteins of pellet (P)
and those of supernatant (S) were subjected to electrophoresis. (A) mtDNAprotein complexes; (B) nDNAprotein complexes. Lane 1; initial
proteins (50 mg). Lanes 2 and 3; proteins and 2 mg DNA. Lanes 4 and 5; proteins and 5 mg DNA. Lanes 6 and 7; proteins and 10 mg DNA.

mitochondrial acid-soluble proteins to DNA showed

that when NaCl concentration increases, the formation of nucleoproteid complexes gradually
decreases to stop completely at 0.5 M NaCl (Fig. 4).
The formation of DNAprotein complexes was
also determined from the capacity of proteins to
compete with actinomycin D for DNA-binding sites.
The binding of actinomycin D to DNA was known to
be accompanied by a change in its optical properties
(Muller and Crothers, 1968). In the formation of a
DNAprotein complex, the binding of actinomycin D
must be lower for the DNAprotein complex than for
free DNA. The absorption spectra of free actinomycin
D, the nDNA-actinomycin D complex and a reconstructed nucleoproteidactinomycin D complex are
presented in Fig. 5A. It appeared that addition of acidsoluble proteins from liver mitochondria to nDNA at
the ratio 1.5:1 reduced actinomycin binding by about
40%, which points to the formation of a DNAprotein

complex. When lDNA was used, a nearly the same

spectrophotometric characteristics of the DNAprotein complex was observed (Fig. 5B). Lambda DNA
from the phage isolated from the E. coli W3110
(c/1857 Sam7) strain consists of 48502 bp, it is nearly
3 times as long as mitochondrial DNA. The study of
the differential spectra of DNA-actinomycin D and
DNAprotein-actinomycin D against actinomycin D
(Fig. 6) showed changes in the character of the
spectral curve for the complex DNAprotein-actinomycin D as compared to the DNA-actinomycin D
complex. These differences are due to different
amounts of actinomycin D bound to free DNA and
the DNAprotein complex. The differential spectra
presented show distinctly that to DNA complexed
with protein a substantially less amount of actinomycin D is bound than to free DNA since the binding
site on DNA in the DNAprotein complex are
partially occupied by protein. Addition of NaCl to

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M.P. Kutsyi et al. / Mitochondrion 5 (2005) 3544

a concentration of 1 M resulted in dissociation of the

DNAprotein complex, which is indicated by a
change in the spectral curve (it tends to became a
direct line). Thus, the results point to that acid-soluble
mitochondrial proteins form complexes with DNA.
3.3. Proteases associated with acid-soluble proteins
of mitochondria. DNA-activated proteases

Fig. 4. Dependence of acid-soluble proteinDNA complex formation

on NaCl concentration. NaCl (0.15 M, 0.35 M, 0.42 M, 0.5 M) and
10 mg nDNA were added to 50 mg of liver mitochondrial proteins in
0.02 M TrisHCl buffer, pH 7.4. After centrifugation, proteins of the
pellet (P) and those of the supernatant (S) were subjected to
electrophoresis. Lane 1; initial proteins (50 mg). Lanes 2 and 3;
proteins and nDNA with 0.15 M NaCl. Lanes 4 and 5; proteins and
nDNA with 0.35 M NaCl. Lanes 6 and 7; proteins and nDNA with
0.42 M NaCl. Lanes 8 and 9; proteins and nDNA with 0.5 M NaCl.

It has been well known that mitochondria contain

many proteases involved in the metabolism of
mitochondrial proteins (Bota and Davies, 2001,
review). In our investigation it was revealed that
along with acid-soluble proteins, also proteases were
extracted that cleaved various polypeptides of these
proteins at various intensity (Fig. 7). We showed for
the first time that the activity of these proteases
increased in the presence of DNA and that denatured
nDNA and mtDNA activated them to a higher degree
than native nDNA and mtDNA do (Fig. 7). The
intensity of cleavage of the acid-soluble proteins by
these proteases was higher than the cleavage of other
mitochondrial proteins. The optimum activity of
proteases associated with acid-soluble proteins was
observed at pH 8.0 (data not shown).
When acid-soluble proteins from liver mitochondria were incubated with the protease inhibitors
antipain, leupeptin, PMSP, NEM, a decrease in
protease activity was observed, which points to the
presence of serine and cysteine proteases (data not
shown).

Fig. 5. Spectrophotometric characteristics of complexes of actinomycin D with DNA and actinomycin D with a nucleoproteid reconstructed
from DNA and acid-soluble proteins of liver mitochondria. (A) liver mitochondrial protein and nDNA. (B) protein and lDNA. 1; free
actinomycin D (50 mg/ml). 2; actinomycin D and DNA (70 mg/ml). 3; actinomycin D and nucleoproteid (protein:DNAZ1.5:1).

M.P. Kutsyi et al. / Mitochondrion 5 (2005) 3544

41

Fig. 6. Differential spectra of DNA-actinomycin D and DNA

protein-actinomycin D against actinomycin D. 1, lDNA-actinomycin D; 2, lDNAprotein-actinomycin D (protein:DNAZ1.5:1); 3,
lDNAprotein-actinomycin D after treatment with 1 M NaCl
(control: actinomycin D and 1 M NaCl).

In order to determine the amount of proteases

associated with acid-soluble proteins and their
molecular masses, these proteins were subjected to
enzyme-electrophoresis in 15% SDS-polyacrylamide
gel containing casein. After electrophoresis, the
gel was incubated as described in Section 2.
After the staining of the gel, proteins and casein
were colored, whereas in the sites of protease location
white spots were formed because of hydrolysis of
acrylamide-bound casein by proteases. In the electrophoregram presented in Fig. 8, seven proteases are
seen associated with acid-soluble proteins of liver and
spleen mitochondria. The proteases have molecular
masses between 20 and 90 kDa. As seen from the
Figure, proteases of mitochondrial acid-soluble proteins of spleen substantially differ from proteases of
those of liver. In particular, in the acid-soluble
proteins from spleen mitochondria a protease of
63 kDa is absent and proteases with molecular masses
33 and 2729 kDa are more active than the same
proteases in the liver mitochondrial proteins.

4. Discussion
Recently a high vulnerability of mtDNA has been
reported by a number of authors. Compared to the
nDNA, the mtDNA is more susceptible not only to
ROS that are formed in mitochondria but also to

Fig. 7. Effect of DNA on the activity of proteases of liver

mitochondrial acid-soluble proteins. Proteins (50 mg) in 0.04 M
TrisHCl buffer, pH 8.0, were incubated for 1.5 h at 37 8C in the
absence and in the presence of denatured or native mtDNA or
nDNA. Lane 1, proteins before incubation; Lane 2, proteins
incubated in the absence of DNA; Lanes 3 and 4, proteins incubated
in the presence of denatured mtDNA and nDNA; Lanes 5 and 6,
proteins incubated in the presence of native mtDNA and nDNA.

various exogenous agents. Exogenous and endogenous factors cause in mtDNA substantially more
lesions than in a nDNA fragment equal in size to
mtDNA (Richter et al., 1988; Richter, 1992; Sawyer
and Van Houten, 1999; Marcelino and Thilly, 1999).
The frequency of mutations and deletions in mtDNA
is 1025 times higher than in nDNA (Ozawa, 1997;
Marcelino and Thilly, 1999). It was suggested that one
of the causes of the high sensitivity of mtDNA to
damaging influences of ROS and other genotoxic
factors is the absence of histones in mitochondria
(Richter, 1992; Wallace, 1992; Higuchi and Linn,
1995). Because of this mitochondrial DNA is not
protected by proteins and does not form a densely
packed nucleoproteid as it is the case in the nuclei.

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M.P. Kutsyi et al. / Mitochondrion 5 (2005) 3544

Fig. 8. Enzyme-electrophoresis of liver and spleen mitochondrial

acid-soluble proteins. After electrophoresis the gel was incubated for
48 h at 37 8C for determination of protease activity. Lane 1, protein
markers; Lane 2, protease activity of liver mitochondrial acidsoluble proteins; Lane 3, protease activity of spleen mitochondrial
acid-soluble proteins.

However, our present study showed that in liver and

spleen mitochondria there are proteins extractable by
0.2 M H2SO4 which is usually used to extract basic
nuclear proteins, histones. These acid-soluble proteins
were represented in liver and spleen mitochondria by
more than 20 polypeptides (Fig. 1). It should be noted
that two of them possessed an electrophoretic
mobility comparable with that of H1 and H2B
histones. Earlier Olszewska and Tait (1980) have
isolated basic proteins similar to histones in electrophoretic mobility from the mitochondrial replication
complex Paramecium aurelia by extraction with
0.25 M HCl. Pavco and Van Tuyle (1985) obtained
from liver mitochondria a histone-like protein P16

binding to mtDNA in the region of the D-loop.

Histone-like proteins were also isolated from mitochondria of yeast and Xenopus laevis (Caron et al.,
1979; Mignotte et al., 1985). Some later, using
chromatography on DEAE-cellulose, Yamada et al.
(1991) isolated histone-like protein HMBH possessing an electrophoretic mobility near to that of histone
H2B from bovine heart mitochondria. The protein
HMBH bears close resemblance to histone-like
protein HM from mitochondria of yeast and some
less resemblance to HU of E. coli, P16 of liver
mitochondria, and mtSSB of Xenopus laevis mitochondria. Yamada et al. (1991) also isolated two
histone-like proteins from an acid extract of bovine
heart mitochondria using chromatography on Bio-Rex
70 and Bio-Gel P-60. One of these proteins also had
an electrophoretic mobility close to that of histone
H2B. Possibly, among the proteins we have isolated
from liver and spleen mitochondria (Fig. 1), there are
histone-like proteins found in the mitochondria of
other cells. It is known that in the presence of 0.2 M
H2SO4, histones, the proteins that possess basic
properties, are extracted from cell nuclei (Bonner et
al., 1968). It can be assumed therefore that a great part
of the proteins we have extracted from mitochondria
are also basic proteins. The acid-soluble mitochondrial proteins we have isolated possess a high
DNA affinity and form nucleoproteid complexes
at the physiological concentration of NaCl. The
DNAprotein complex formation ceases completely
on increasing the NaCl concentration up to 0.5 M. It
has been known that dissociation of the linker histone
H1 from thymus nucleohistones occurs at about
0.40.5 M NaCl (Bonner et al., 1968).Thus, acidsoluble proteins of mitochondria behave in the same
manner as histones with respect to forming DNA
protein complexes. Possibly, these proteins fulfil in
mitochondria the functions of histones. Recently it has
been found that in mammalian mitochondria there are
RNA-binding proteins which are likely to be involved
in the initiation of transcription. It should be noted
that two of these proteins are bound to the inner
mitochondrial membrane (Koc and Spremulli, 2003).
It has been known that in mammalian mitochondria
there are DNA-binding proteins involved in mtDNA
functioning. These are transcription factor 1 (mtTF1),
transcription factor A of mitochondria (TFAM),
and polymerase gamma (POLG) (Fisher et al., 1991;

M.P. Kutsyi et al. / Mitochondrion 5 (2005) 3544

Garrido et al., 2003). Recently it has been shown that

an ATP-dependent protease, Lon protease, an analoque of La protease (product of the Lon gene) from
E. coli is present in the mitochondria of various
mammalian tissues (Wang et al., 1993). The presence
of numerous proteases in mitochondria has been well
known (Bota and Davies, 2001, review). Lon protease
is capable of binding to mtDNA and seems to
participate in the degradation of proteins controlling
mtDNA replication and gene expression (Fu and
Markovitz, 1998). In our studies we found that,
together with acid-soluble proteins of mitochondria,
also proteases are isolated that cleave different
fractions of these proteins at different intensities.
We have shown for the first time that proteases
isolated together with mitochondrial acid-soluble
proteins are activated by DNA. The functions of
these proteases have not yet been studied. It is
possible that proteases we have found participate in
the control of mtDNA functioning, like Lon protease.
Earlier we detected proteases tightly bound to thymus
nuclear histones. These proteases are H1 histonespecific and are activated by DNA containing breaks
or denatured regions (Kutsyi and Gaziev, 1994). The
activity of histone-associated proteases considerably
increases in the nuclei of a regenerating rat liver in the
period of intensive transcription and replication of
DNA as well as in thymus nuclei upon various
influences leading to DNA degradation (exposure to
g-radiation, hydrocortisone injection) (Kutsyi et al.,
1992; Kutsyi and Gaziev, 1994). When histoneassociated proteases cleave histone H1 in the nuclei,
they participate in the decompaction of the highly
ordered structure of chromatin and thus facilitate the
access of enzymes of transcription, replication, repair
or degradation to DNA. Possibly, DNA-dependent
proteases extracted together with acid-soluble proteins of mitochondria provide access of various
enzymes to mtDNA thus being involved in mtDNA
replication, transcription, repair, and degradation. The
ability of these proteases to bind to DNA and their
activation by DNA, especially by a denatured DNA
seems to point to the participation of these enzymes in
the functioning of mtDNA.
At present, it is absolutely unclear what is the role
of acid-soluble proteins we have detected and their
accompanying proteases including DNA-activated
ones in the functioning of mitochondria and

43

mtDNA. Because mitochondrial acid-soluble proteins, like histones, are capable of binding to DNA
and forming nucleoproteid complexes it may be
assumed that these proteins perform in the mitochondria a function similar to that of histones in the nuclei.
When binding to mtDNA, acid-soluble proteins
probably influence the structural organization and
the functional activity of mtDNA. Because proteases
that are isolated together with acid-soluble proteins
intensively cleave these proteins it can be suggested
that DNA, via DNA-activated proteases, can regulate
the number of basic proteins and thus control its own
structural organization and functional activity. It is
also probable that DNA-activated proteases are
involved in mtDNA replication and transcription,
while cleaving repressor proteins. It is suggested that
mitochondria in which DNA is severely damaged
undergo elimination. It is quite possible that the DNAactivated proteases we have detected participate in the
elimination of mitochondria, the process triggered by
damaged mtDNA.

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