Abstract
A high molecular weight mitochondrial DNA (mtDNA) replication complex, associated with the mitochondrial membrane, was isolated by sucrose gradient centrifugation from purified wheat embryo mitochondria. This complex comprised the mtDNA as well as enzyme activities involved in the replication and transcription of the organelle genome, such as DNA polymerase, RNA polymerase and topoisomerase type I. The isolated complex is active in mtDNA and mtRNA synthesis in vitro. Electron microscopy and lipid analysis confirmed the membrane origin of this complex. Enzyme activities are resistant to physiological ionic strengths, 0.1–0.2 M KCl, while the membrane-mtDNA association is resistant up to 1 M KCl. DNase treatment of the complex released the DNA polymerase activity while protease treatment solubilized mtDNA, suggesting the direct interaction of mtDNA with membrane protein(s). The use of a novel approach to detect mtDNA fragments specifically retained by the mitochondrial membranes after Sal I digestion of the complex suggests that specific mtDNA sequences anchor mtDNA to mitochondrial membranes.
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Abbott AG, O'Dell M, Flavell RB: Quantitative variation in components of the maize mitochondrial genome between tissues and between plants with different male-sterile cytoplasms. Plant Mol Biol 4: 233–240 (1985).
Bailey-Serres J, Leroy P, Jones SS, Wahleithner JA, Wolstenholme DR: Size distributions of circular molecules in plant mitochondrial DNAs. Curr Genet 12: 49–53 (1987).
Baldacci G, Chérif-Zahar B, Bernardi G: The initiation of DNA replication in the mitochondrial genome of yeast. EMBO J 3: 2115–2120 (1984).
Barat M, Rickwood D, Dufresne C, Mounolou JC: Characterization of DNA-protein complexes from the mitochondria of Xenopus laevis oocytes. Exp Cell Res 157: 207–217 (1985).
Bedinger P, Walbot V: DNA synthesis in purified maize mitochondria. Curr Genet 10: 631–637 (1986).
Brown GL, Kemble RJ: An in vitro DNA replication complex from Brassica mitochondria. Curr Genet 15: 341–347 (1989).
Christophe L, Tarrago-Litvak L, Castroviejo M, Litvak S: Mitochondrial DNA polymerase from wheat embryos. Plant Sci Lett 21: 181–192 (1981).
Clayton D: Replication of animal mitochondrial DNA. Cell 28: 693–705 (1982).
Chang DD, Hauswirth WW, Clayton DA: Replication priming and transcription initiate from precisely the same site in mouse mitochondrial DNA. EMBO J 4: 1559–1567 (1985).
DeFrancesco L, Attardi G: In situ photochemical cross-linking of HeLa cell mitochondrial DNA by a psoralen derivative reveals a protected region near the origin of replication. Nucl Acids Res 9: 6017–6030 (1981).
Douce R: Mitochondria in higher plants: structure, function and biogenesis. Academic Press, London (1985).
Echeverria M, Martin MT, Ricard B, Litvak S: A DNA topoisomerase type I from wheat embryo mitochondria. Plant Mol Biol 6: 417–427 (1986).
Echeverria M, Robert D, Carde JP, Litvak S: In: Quetier F, Lejeune B (eds) Proceedings of the 3rd International Workshop on the Mitochondrial Genome of Higher Plants, 28 August to 2 September, Roscoff, France, p. 20. C.N.R.S. France (1988).
Heape AM, Juguelin H, Boiron F, Cassagne C: Improved one-dimensional thin-layer chromatographic technique for polar lipids. J Chromatogr 322: 391–395 (1985).
Hoshino T, McKenzie T, Schmidt S, Tanaka T, Sueoka N: Nucleotide sequence of Bacillus subtilis dnaB: a gene essential for DNA replication initiation and membrane attachment. Proc Natl Acad Sci USA 84: 653–657 (1987).
Jackson DA, Cook PR: Replication occurs at a nucleoskeleton. EMBO J 5: 1403–1410 (1986).
Kawano S, Kuroiwa T: Isolation and characterization of a membrane-DNA complex in the mitochondria of Physarum polycephalum. Exp Cell Res 161: 460–472 (1985).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lowry M, Roseborough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent J Biol Chem 193: 265–275 (1951).
Martin MT, Echeverria M, Litvak S, Araya A: Studies of transcription in isolated wheat mitochondria and organelle extracts. Plant Sci 49: 199–207 (1987).
Mignotte B, Barat M, Mounolou JC: Characterization of a mitochondrial protein binding to single stranded DNA. Nucl Acids Res 13: 1703–1716 (1985).
Nass MJK: Mitochondrial DNA: intramitochondrial distribution and structural relations of single and double length circular DNA. J Mol Biol 42: 521–528 (1969).
Ozias-Akins P, Tabaeizadeh Z, Pring DR, Vasil I: Preferential amplification of mitochondrial DNA fragments in somatic hybrid of the Gramineae. Curr Genet 13: 241–245 (1988).
Pabo CO, Sauer RT: Protein-DNA recognition. Ann Rev Biochem 53: 293–321 (1984).
Pardue ML, Foster JM, Cech TR: DNA-protein interaction in the Drosophila virilis mitochondrial chromosome. Nucl Acids Res 12: 1991–1999 (1984).
Palmer JP, Shields CR: Tripartite structure of the Bassica campestris mitochondrial genome. Nature 307: 437–440 (1982).
Pring DR, Lonsdale DM: Molecular biology of higher plant mitochondrial DNA. Int Rev Cytol 97: 1–46 (1985).
Quetier F, Lejeune B, Delorme S, Falconet D: Molecular organzation and expression of the mitochondrial genome of higher plants. In: Douce R, Day DA (eds) Encyclopaedia of Plant Physiology Springer-Verlag, Berlin (1985).
Ricard B, Echeverria M, Christophe L, Litvak S: DNA synthesis in isolated mitochondria and mitochondrial extracts from wheat embryos. Plant Mol Biol 2: 167–175 (1983).
Shearman CW, Kalf GF: DNA replication by a membrane-DNA complex from rat liver mitochondria. Arch Biochem Biophys 182: 573–582 (1977).
VanTuyle GC, Pavco PA: The rat liver mitochondria DNA-protein complex: displaced single-strands of replicative intermediates are protein-coated. J Cell Biol 100: 251–257 (1985).
Yung BY, Kornberg A: Membrane attachment activates dnaA protein, the initiation protein of chromosome replication in Escherichia coli. Proc Natl Acad Sci USA 85: 7202–7205 (1988).
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Echeverria, M., Robert, D., Carde, J.P. et al. Isolation from wheat mitochondria of a membrane-associated high molecular weight complex involved in DNA synthesis. Plant Mol Biol 16, 301–315 (1991). https://doi.org/10.1007/BF00020561
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DOI: https://doi.org/10.1007/BF00020561