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. 1995 Jan;15(1):580–589. doi: 10.1128/mcb.15.1.580

A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence.

B Xu 1, D A Clayton 1
PMCID: PMC232017  PMID: 7528331

Abstract

Critical features of the mitochondrial leading-strand DNA replication origin are conserved from Saccharomyces cerevisiae to humans. These include a promoter and a downstream GC-rich sequence block (CSBII) that encodes rGs within the primer RNA. During in vitro transcription at yeast mitochondrial replication origins, there is stable and persistent RNA-DNA hybrid formation that begins at the 5' end of the rG region. The short rG-dC sequence is the necessary and sufficient nucleic acid element for establishing stable hybrids, and the presence of rGs within the RNA strand of the RNA-DNA hybrid is required. The efficiency of hybrid formation depends on the length of RNA synthesized 5' to CSBII and the type of RNA polymerase employed. Once made, the RNA strand of an RNA-DNA hybrid can serve as an effective primer for mitochondrial DNA polymerase. These results reveal a new mechanism for persistent RNA-DNA hybrid formation and suggest a step in priming mitochondrial DNA replication that requires both mitochondrial RNA polymerase and an rG-dC sequence-specific event to form an extensive RNA-DNA hybrid.

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Selected References

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  1. Blanc H., Dujon B. Replicator regions of the yeast mitochondrial DNA responsible for suppressiveness. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3942–3946. doi: 10.1073/pnas.77.7.3942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang D. D., Clayton D. A. A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA. Science. 1987 Mar 6;235(4793):1178–1184. doi: 10.1126/science.2434997. [DOI] [PubMed] [Google Scholar]
  3. Chang D. D., Clayton D. A. A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication. EMBO J. 1987 Feb;6(2):409–417. doi: 10.1002/j.1460-2075.1987.tb04770.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang D. D., Clayton D. A. Priming of human mitochondrial DNA replication occurs at the light-strand promoter. Proc Natl Acad Sci U S A. 1985 Jan;82(2):351–355. doi: 10.1073/pnas.82.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang D. D., Hauswirth W. W., Clayton D. A. Replication priming and transcription initiate from precisely the same site in mouse mitochondrial DNA. EMBO J. 1985 Jun;4(6):1559–1567. doi: 10.1002/j.1460-2075.1985.tb03817.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Christianson T. W., Clayton D. A. A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro. Mol Cell Biol. 1988 Oct;8(10):4502–4509. doi: 10.1128/mcb.8.10.4502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clayton D. A. Replication and transcription of vertebrate mitochondrial DNA. Annu Rev Cell Biol. 1991;7:453–478. doi: 10.1146/annurev.cb.07.110191.002321. [DOI] [PubMed] [Google Scholar]
  8. Côté J., Renaud J., Ruiz-Carrillo A. Recognition of (dG)n.(dC)n sequences by endonuclease G. Characterization of the calf thymus nuclease. J Biol Chem. 1989 Feb 25;264(6):3301–3310. [PubMed] [Google Scholar]
  9. Côté J., Ruiz-Carrillo A. Primers for mitochondrial DNA replication generated by endonuclease G. Science. 1993 Aug 6;261(5122):765–769. doi: 10.1126/science.7688144. [DOI] [PubMed] [Google Scholar]
  10. Dasgupta S., Masukata H., Tomizawa J. Multiple mechanisms for initiation of ColE1 DNA replication: DNA synthesis in the presence and absence of ribonuclease H. Cell. 1987 Dec 24;51(6):1113–1122. doi: 10.1016/0092-8674(87)90597-6. [DOI] [PubMed] [Google Scholar]
  11. Fangman W. L., Henly J. W., Brewer B. J. RPO41-independent maintenance of [rho-] mitochondrial DNA in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Jan;10(1):10–15. doi: 10.1128/mcb.10.1.10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fangman W. L., Henly J. W., Churchill G., Brewer B. J. Stable maintenance of a 35-base-pair yeast mitochondrial genome. Mol Cell Biol. 1989 May;9(5):1917–1921. doi: 10.1128/mcb.9.5.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Foury F. Cloning and sequencing of the nuclear gene MIP1 encoding the catalytic subunit of the yeast mitochondrial DNA polymerase. J Biol Chem. 1989 Dec 5;264(34):20552–20560. [PubMed] [Google Scholar]
  14. Foury F., Vanderstraeten S. Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity. EMBO J. 1992 Jul;11(7):2717–2726. doi: 10.1002/j.1460-2075.1992.tb05337.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuller C. W., Richardson C. C. Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Site and direction of initial DNA synthesis. J Biol Chem. 1985 Mar 10;260(5):3185–3196. [PubMed] [Google Scholar]
  16. Gillum A. M., Clayton D. A. Mechanism of mitochondrial DNA replication in mouse L-cells: RNA priming during the initiation of heavy-strand synthesis. J Mol Biol. 1979 Dec 5;135(2):353–368. doi: 10.1016/0022-2836(79)90441-8. [DOI] [PubMed] [Google Scholar]
  17. Greenleaf A. L., Kelly J. L., Lehman I. R. Yeast RPO41 gene product is required for transcription and maintenance of the mitochondrial genome. Proc Natl Acad Sci U S A. 1986 May;83(10):3391–3394. doi: 10.1073/pnas.83.10.3391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Itoh T., Tomizawa J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A. 1980 May;77(5):2450–2454. doi: 10.1073/pnas.77.5.2450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Karwan R., Bennett J. L., Clayton D. A. Nuclear RNase MRP processes RNA at multiple discrete sites: interaction with an upstream G box is required for subsequent downstream cleavages. Genes Dev. 1991 Jul;5(7):1264–1276. doi: 10.1101/gad.5.7.1264. [DOI] [PubMed] [Google Scholar]
  20. Kelly J. L., Lehman I. R. Yeast mitochondrial RNA polymerase. Purification and properties of the catalytic subunit. J Biol Chem. 1986 Aug 5;261(22):10340–10347. [PubMed] [Google Scholar]
  21. Kiss T., Filipowicz W. Evidence against a mitochondrial location of the 7-2/MRP RNA in mammalian cells. Cell. 1992 Jul 10;70(1):11–16. doi: 10.1016/0092-8674(92)90528-k. [DOI] [PubMed] [Google Scholar]
  22. Li K., Smagula C. S., Parsons W. J., Richardson J. A., Gonzalez M., Hagler H. K., Williams R. S. Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis. J Cell Biol. 1994 Mar;124(6):871–882. doi: 10.1083/jcb.124.6.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lisowsky T., Michaelis G. A nuclear gene essential for mitochondrial replication suppresses a defect of mitochondrial transcription in Saccharomyces cerevisiae. Mol Gen Genet. 1988 Oct;214(2):218–223. doi: 10.1007/BF00337714. [DOI] [PubMed] [Google Scholar]
  24. Low R. L., Cummings O. W., King T. C. The bovine mitochondrial endonuclease prefers a conserved sequence in the displacement loop region of mitochondrial DNA. J Biol Chem. 1987 Nov 25;262(33):16164–16170. [PubMed] [Google Scholar]
  25. Masters B. S., Stohl L. L., Clayton D. A. Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7. Cell. 1987 Oct 9;51(1):89–99. doi: 10.1016/0092-8674(87)90013-4. [DOI] [PubMed] [Google Scholar]
  26. Masukata H., Tomizawa J. A mechanism of formation of a persistent hybrid between elongating RNA and template DNA. Cell. 1990 Jul 27;62(2):331–338. doi: 10.1016/0092-8674(90)90370-t. [DOI] [PubMed] [Google Scholar]
  27. Roberts R. W., Crothers D. M. Stability and properties of double and triple helices: dramatic effects of RNA or DNA backbone composition. Science. 1992 Nov 27;258(5087):1463–1466. doi: 10.1126/science.1279808. [DOI] [PubMed] [Google Scholar]
  28. Schinkel A. H., Groot Koerkamp M. J., Tabak H. F. Mitochondrial RNA polymerase of Saccharomyces cerevisiae: composition and mechanism of promoter recognition. EMBO J. 1988 Oct;7(10):3255–3262. doi: 10.1002/j.1460-2075.1988.tb03192.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schinkel A. H., Tabak H. F. Mitochondrial RNA polymerase: dual role in transcription and replication. Trends Genet. 1989 May;5(5):149–154. doi: 10.1016/0168-9525(89)90056-5. [DOI] [PubMed] [Google Scholar]
  30. Schmitt M. E., Clayton D. A. Conserved features of yeast and mammalian mitochondrial DNA replication. Curr Opin Genet Dev. 1993 Oct;3(5):769–774. doi: 10.1016/s0959-437x(05)80097-8. [DOI] [PubMed] [Google Scholar]
  31. Schmitt M. E., Clayton D. A. Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Dec;13(12):7935–7941. doi: 10.1128/mcb.13.12.7935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schmitt M. E., Clayton D. A. Yeast site-specific ribonucleoprotein endoribonuclease MRP contains an RNA component homologous to mammalian RNase MRP RNA and essential for cell viability. Genes Dev. 1992 Oct;6(10):1975–1985. doi: 10.1101/gad.6.10.1975. [DOI] [PubMed] [Google Scholar]
  33. Stohl L. L., Clayton D. A. Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming. Mol Cell Biol. 1992 Jun;12(6):2561–2569. doi: 10.1128/mcb.12.6.2561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Topper J. N., Bennett J. L., Clayton D. A. A role for RNAase MRP in mitochondrial RNA processing. Cell. 1992 Jul 10;70(1):16–20. doi: 10.1016/0092-8674(92)90529-l. [DOI] [PubMed] [Google Scholar]
  35. Wettstein-Edwards J., Ticho B. S., Martin N. C., Najarian D., Getz G. S. In vitro transcription and promoter strength analysis of five mitochondrial tRNA promoters in yeast. J Biol Chem. 1986 Feb 25;261(6):2905–2911. [PubMed] [Google Scholar]
  36. Xu B., Clayton D. A. Assignment of a yeast protein necessary for mitochondrial transcription initiation. Nucleic Acids Res. 1992 Mar 11;20(5):1053–1059. doi: 10.1093/nar/20.5.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zassenhaus H. P., Hofmann T. J., Uthayashanker R., Vincent R. D., Zona M. Construction of a yeast mutant lacking the mitochondrial nuclease. Nucleic Acids Res. 1988 Apr 25;16(8):3283–3296. doi: 10.1093/nar/16.8.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. de Zamaroczy M., Faugeron-Fonty G., Baldacci G., Goursot R., Bernardi G. The ori sequences of the mitochondrial genome of a wild-type yeast strain: number, location, orientation and structure. Gene. 1984 Dec;32(3):439–457. doi: 10.1016/0378-1119(84)90019-2. [DOI] [PubMed] [Google Scholar]

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