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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
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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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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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.
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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).
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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
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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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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