Summary
To study the structure of in vivo mitochondrial DNA recombination intermediates in Saccharomyces cerevisiae, we used a deletion mutant of the wild type mitochondrial genome. The mtDNA of this petite is composed of a direct tandem repetition of an ∼4,600 pb monomer repeat unit with a unique HhaI restriction enzyme site per repeat. The structure of native mtDNA isolated from log phase cells, and mtDNA crosslinked in vivo with trioxsalen plus UVA irradiation, was studied by electron microscopy. Both populations contained crossed strand “Holliday” type recombination intermediates. Digestion of both non-crosslinked and crosslinked and mtDNA with the enzyme HhaI released X and H shaped structures composed of two monomers. Electron microscopic analysis revealed that these structures had pairs of equal length arms as required for homologous recombination intermediates and that junctions could occur at points along the entire monomer length. The percentage of recombining monomers in both non-crosslinked and trioxsalen crosslinked mtDNA was calculated by quantitative analysis of all the structures present in an HhaI digest. The relationship between these values and the apparent dispersive replication of mtDNA in density-shift experiments and mtDNA fragility during isolation is discussed.
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References
Bell L, Byers B (1979) Occurrence of crossed strand-exchange forms in yeast DNA during meiosis. Proc Natl Acad Sci USA 76:3445–3449
Broker TR, Lehman IR (1971) Branched DNA molecules: Intermediates in T4 recombination. J mol Biol 60:131–149
Cech TR, Pardue ML (1976) Electron microscopy of DNA cross-linked with trimethylpsoralen: Test of the secondary structure of eucaryotic inverted repeat sequences. Proc Natl Acad Sci USA 73:2644–2648
Clayton DA (1982) Replication of animal mitochondrial DNA. Cell 28:693–705
Davis RW, Simon M, Davidson N (1971) Electron microscopic heteroduplex method for mapping regions of base sequence homology in nucleic acids. 21:413–418
Dujon B (1981) Mitochondrial genetics and functions. In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast Saccharomyces: life cycle and inheritance. Cold Spring Harbor Laboratory, New York, p 505–635
Goddard JM, Wolstenholme DR (1978) Origin and direction of replication in mitochondrial DNA molecules from Drosophila melanogaster. Proc Natl Acad Sci USA 75:3886–3890
Hajduk SL, Klein VA, Englund PT (1984) Replication of kinetoplast DNA maxicircles. Cell 36:483–492
Hollenberg CP, Borst P, Van Bruggen EFJ (1970) Mitochondrial DNA V. A 25 μm closed circular duplex DNA molecule in wild-type yeast mitochondria. Structure and genetic complexity. Biochem Biophys Acta 209:1–15
Holliday R (1964) A mechanism for gene conversion in fungi. Genet Res 5:282–304
Jacquier A, Dujon B (1985) An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene. Cell 41:383–394
Johnston LH, Williamson DH (1978) An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae. Mol Gen Genet 164:217–225
Kozinski AW (1961) Fragmentary transfer of 32P-labelled parental DNA to progeny phage. Virology 13:124–134
Locker J, Rabinowitz M, Getz GS (1974) Electron microscopic and renaturation kinetic analysis of mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae. J Mol Biol 88:489–507
Luder A, Mosig G (1983) Two alternative mechanisms for initiation of DNA replication forks in bacteriophage T4: priming by RNA polymerase and by recombination. Proc Natl Acad Sci USA 79:1101–1105
Macreadie IG, Scott RM, Zinn AR, Butow RA (1985) Transposition of an intron in yeast mitochondria requires a protein encoded by that intron. Cell 41:395–402
Meselson M, Stahl FW (1958) The replication of DNA in Escherichia coli. Proc Natl Acad Sci USA 44:671–682
Meselson MS, Radding CM (1975) A general model for genetic recombination. Proc Natl Acad Sci USA 72:358–361
Michaelis G, Petrochilo E, Slonimski PP (1973) Mitochondrial genetics III. Recombined molecules of mitochondrial DNA obtained from crosses between cytoplasmic petite mutants of Saccharomyces cerevisiae: physical and genetic characterization. Mol Gen Genet 123:51–65
Potter H, Dressler D (1976) On the mechanism of genetic recombination: Electron microscopic observation of recombination intermediates. Proc Natl Acad Sci USA 73:3000–3004
Praskash L (1975) Repair of pyrimidine dimers in nuclear and mitochondrial DNA of yeast irradiated with low doses of ultraviolet light. J Mol Biol 98:781–795
Sena EP (1982) Effects of the kar gene on cytoplasmic mixing and mitochondrial genome suppressiveness, and consequences for cytoduction of petite DNA in Saccharomyces cerevisiae. Curr Genet 5:47–52
Sena EP, Welch J, Halvorson HO, Fogel S (1975) Nuclear and mitochondrial deoxyribonucleic acid replication during mitosis in Saccharomyces cerevisiae. J Bacteriol 123:497–504
Sigal N, Alberts B (1972) Genetic recombination: The nature of a crossed strand-exchange between two homologous DNA molecules. J Mol Biol 71:789–793
Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW (1983) The double-strand-break-repair model for recombination. Cell 33:25–35
Tomizawa JI, Anraku N (1964) Molecular mechanisms of genetic recombination in bacteriophage II: Joining of parental DNA molecules of phage T4. J Mol Biol 8:516–540
Valenzuela MS, Inman RB (1975) Visualization of a novel junction in bacteriophage λ DNA. Proc Natl Acad Sci USA 72:3024–3028
Van Winkle-Swift KP, Birky CW Jr (1978) The non-reciprocality of organelle gene recombination in Chlamydomonas reinhardtii and Saccharomyces cerevisiae. Mol Gen Genet 166:193–209
Warner RC, Fishel RA, Wheeler FC (1979) Branch migration in recombination. Cold Spring Harbor Symp Quant Biol 43:957–968
Waters R, Moustacchi E (1974) The disappearance of ultraviolet-induced pyrimidine dimers from the nuclear DNA of exponential and stationary phase cells of Saccharomyces cerevisiae following various post-irradiation treatments. Biochim Biophys Acta 366:241–250
Willamson DH, Fennell DJ (1974) Apparent dispersive replication of yeast mitochondrial DNA as revealed by density labelling experiments. Mol Gen Genet 131:193–207
Williamson DH, Moustacchi E (1971) The synthesis of mitochondrial DNA in synchronously dividing cultures of Saccharomyces cerevisiae. Biochem Biophys Res Commun 42:195–201
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Communicated by W. Gajewski
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Sena, E.P., Revet, B. & Moustacchi, E. In vivo homologous recombination intermediates of yeast mitochondrial DNA analyzed by electron microscopy. Molec Gen Genet 202, 421–428 (1986). https://doi.org/10.1007/BF00333272
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DOI: https://doi.org/10.1007/BF00333272