Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Mar;11(3):1696–1706. doi: 10.1128/mcb.11.3.1696

The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase.

U Schulte 1, A M Lambowitz 1
PMCID: PMC369474  PMID: 1705012

Abstract

The LaBelle-1b strain of Neurospora intermedia contains a 4.1-kb closed-circular mitochondrial plasmid DNA, which encodes a single long open reading frame of 1,151 amino acids reported to have sequence similarity to reverse transcriptases. Here, we show that the LaBelle strain contains a novel DNA polymerase activity that is highly specific for the endogenous LaBelle plasmid DNA in nucleoprotein particles and can be distinguished from the mitochondrial DNA polymerase by several characteristics. Photolabeling experiments indicate that the LaBelle-specific DNA polymerase activity is associated with a polypeptide of 120 kDa, which is in good agreement with the size predicted for the protein encoded by the LaBelle plasmid open reading frame (132 kDa). This 120-kDa polypeptide is found only in the LaBelle strain that contains the mitochondrial plasmid, and it cosegregates with mitochondria in sexual crosses, suggesting that it is encoded by the plasmid. The LaBelle-specific DNA polymerase efficiently uses the artificial DNA substrates, poly(dA)-oligo(dT) and poly(dC)-oligo(dG), but despite its reported sequence similarity to reverse transcriptases, it has very low activity with analogous RNA substrates, poly(rA)-oligo(dT), poly(rC)-oligo(dG), or poly(rCm)-oligo(dG). Considered together with the previous sequence comparisons, our results suggest that the LaBelle plasmid encodes a novel DNA polymerase, which was derived from a protein that was at one time a reverse transcriptase but lost its ability to use RNA templates. This DNA polymerase now presumably functions in replication of the plasmid. Our results constitute the first biochemical evidence for a DNA polymerase activity associated with a mitochondrial plasmid. Further, they may provide insight into the evolution of DNA polymerases from reverse transcriptases, as presumably occurred in the course of evolution following the transition from the so-called RNA world to the present DNA world.

Full text

PDF
1696

Images in this article

1700Image
on p.1700
1700Image
on p.1700
1701Image
on p.1701
1703Image
on p.1703
1704Image
on p.1704

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akins R. A., Grant D. M., Stohl L. L., Bottorff D. A., Nargang F. E., Lambowitz A. M. Nucleotide sequence of the Varkud mitochondrial plasmid of Neurospora and synthesis of a hybrid transcript with a 5' leader derived from mitochondrial RNA. J Mol Biol. 1988 Nov 5;204(1):1–25. doi: 10.1016/0022-2836(88)90594-3. [DOI] [PubMed] [Google Scholar]
  2. Akins R. A., Kelley R. L., Lambowitz A. M. Characterization of mutant mitochondrial plasmids of Neurospora spp. that have incorporated tRNAs by reverse transcription. Mol Cell Biol. 1989 Feb;9(2):678–691. doi: 10.1128/mcb.9.2.678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Akins R. A., Lambowitz A. M. The [poky] mutant of Neurospora contains a 4-base-pair deletion at the 5' end of the mitochondrial small rRNA. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3791–3795. doi: 10.1073/pnas.81.12.3791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Argos P. A sequence motif in many polymerases. Nucleic Acids Res. 1988 Nov 11;16(21):9909–9916. doi: 10.1093/nar/16.21.9909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernad A., Blanco L., Lázaro J. M., Martín G., Salas M. A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell. 1989 Oct 6;59(1):219–228. doi: 10.1016/0092-8674(89)90883-0. [DOI] [PubMed] [Google Scholar]
  6. Bernad A., Zaballos A., Salas M., Blanco L. Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases. EMBO J. 1987 Dec 20;6(13):4219–4225. doi: 10.1002/j.1460-2075.1987.tb02770.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chow T. Y., Fraser M. J. Purification and properties of single strand DNA-binding endo-exonuclease of Neurospora crassa. J Biol Chem. 1983 Oct 10;258(19):12010–12018. [PubMed] [Google Scholar]
  8. Collins R. A., Lambowitz A. M. Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature. Plasmid. 1983 Jan;9(1):53–70. doi: 10.1016/0147-619x(83)90031-8. [DOI] [PubMed] [Google Scholar]
  9. Collins R. A., Stohl L. L., Cole M. D., Lambowitz A. M. Characterization of a novel plasmid DNA found in mitochondria of N. crassa. Cell. 1981 May;24(2):443–452. doi: 10.1016/0092-8674(81)90335-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Garriga G., Bertrand H., Lambowitz A. M. RNA splicing in Neurospora mitochondria: nuclear mutants defective in both splicing and 3' end synthesis of the large rRNA. Cell. 1984 Mar;36(3):623–634. doi: 10.1016/0092-8674(84)90342-8. [DOI] [PubMed] [Google Scholar]
  12. Garriga G., Lambowitz A. M. Protein-dependent splicing of a group I intron in ribonucleoprotein particles and soluble fractions. Cell. 1986 Aug 29;46(5):669–680. doi: 10.1016/0092-8674(86)90342-9. [DOI] [PubMed] [Google Scholar]
  13. Gerard G. F. Poly (2'O-methylcytidylate).oligodeoxyguanylate, a template-primer specific for reverse transcriptase, is not utilized by HeLa cell gamma DNA polymerases. Biochem Biophys Res Commun. 1975 Apr 7;63(3):706–711. doi: 10.1016/s0006-291x(75)80441-4. [DOI] [PubMed] [Google Scholar]
  14. Insdorf N. F., Bogenhagen D. F. DNA polymerase gamma from Xenopus laevis. I. The identification of a high molecular weight catalytic subunit by a novel DNA polymerase photolabeling procedure. J Biol Chem. 1989 Dec 25;264(36):21491–21497. [PubMed] [Google Scholar]
  15. Kamer G., Argos P. Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res. 1984 Sep 25;12(18):7269–7282. doi: 10.1093/nar/12.18.7269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuiper M. T., Lambowitz A. M. A novel reverse transcriptase activity associated with mitochondrial plasmids of Neurospora. Cell. 1988 Nov 18;55(4):693–704. doi: 10.1016/0092-8674(88)90228-0. [DOI] [PubMed] [Google Scholar]
  17. Kuiper M. T., Sabourin J. R., Lambowitz A. M. Identification of the reverse transcriptase encoded by the Mauriceville and Varkud mitochondrial plasmids of Neurospora. J Biol Chem. 1990 Apr 25;265(12):6936–6943. [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lambowitz A. M., Akins R. A., Kelley R. L., Pande S., Nargang F. E. Mitochondrial plasmids of Neurospora and other filamentous fungi. Basic Life Sci. 1986;40:83–92. doi: 10.1007/978-1-4684-5251-8_7. [DOI] [PubMed] [Google Scholar]
  20. Lambowitz A. M., LaPolla R. J., Collins R. A. Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins. J Cell Biol. 1979 Jul;82(1):17–31. doi: 10.1083/jcb.82.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lambowitz A. M. Preparation and analysis of mitochondrial ribosomes. Methods Enzymol. 1979;59:421–433. doi: 10.1016/0076-6879(79)59103-4. [DOI] [PubMed] [Google Scholar]
  22. Meinhardt F., Kempken F., Kämper J., Esser K. Linear plasmids among eukaryotes: fundamentals and application. Curr Genet. 1990 Feb;17(2):89–95. doi: 10.1007/BF00312851. [DOI] [PubMed] [Google Scholar]
  23. Michel F., Lang B. F. Mitochondrial class II introns encode proteins related to the reverse transcriptases of retroviruses. Nature. 1985 Aug 15;316(6029):641–643. doi: 10.1038/316641a0. [DOI] [PubMed] [Google Scholar]
  24. Nargang F. E., Bell J. B., Stohl L. L., Lambowitz A. M. The DNA sequence and genetic organization of a Neurospora mitochondrial plasmid suggest a relationship to introns and mobile elements. Cell. 1984 Sep;38(2):441–453. doi: 10.1016/0092-8674(84)90499-9. [DOI] [PubMed] [Google Scholar]
  25. Natvig D. O., May G., Taylor J. W. Distribution and evolutionary significance of mitochondrial plasmids in Neurospora spp. J Bacteriol. 1984 Jul;159(1):288–293. doi: 10.1128/jb.159.1.288-293.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pande S., Lemire E. G., Nargang F. E. The mitochondrial plasmid from Neurospora intermedia strain Labelle-1b contains a long open reading frame with blocks of amino acids characteristic of reverse transcriptases and related proteins. Nucleic Acids Res. 1989 Mar 11;17(5):2023–2042. doi: 10.1093/nar/17.5.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Poch O., Sauvaget I., Delarue M., Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989 Dec 1;8(12):3867–3874. doi: 10.1002/j.1460-2075.1989.tb08565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stark M. J., Mileham A. J., Romanos M. A., Boyd A. Nucleotide sequence and transcription analysis of a linear DNA plasmid associated with the killer character of the yeast Kluyveromyces lactis. Nucleic Acids Res. 1984 Aug 10;12(15):6011–6030. doi: 10.1093/nar/12.15.6011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stohl L. L., Akins R. A., Lambowitz A. M. Characterization of deletion derivatives of an autonomously replicating Neurospora plasmid. Nucleic Acids Res. 1984 Aug 10;12(15):6169–6178. doi: 10.1093/nar/12.15.6169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stohl L. L., Collins R. A., Cole M. D., Lambowitz A. M. Characterization of two new plasmid DNAs found in mitochondria of wild-type Neurospora intermedia strains. Nucleic Acids Res. 1982 Mar 11;10(5):1439–1458. doi: 10.1093/nar/10.5.1439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stohl L. L., Lambowitz A. M. Construction of a shuttle vector for the filamentous fungus Neurospora crassa. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1058–1062. doi: 10.1073/pnas.80.4.1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Taylor J. W., Smolich B. D., May G. An evolutionary comparison of homologous mitochondrial plasmid DNAs from three Neurospora species. Mol Gen Genet. 1985;201(2):161–167. doi: 10.1007/BF00425654. [DOI] [PubMed] [Google Scholar]
  33. Uyemura D., Lehman I. R. Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. I. The polA12 mutation. J Biol Chem. 1976 Jul 10;251(13):4078–4084. [PubMed] [Google Scholar]
  34. Xiong Y., Eickbush T. H. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 1990 Oct;9(10):3353–3362. doi: 10.1002/j.1460-2075.1990.tb07536.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Xiong Y., Eickbush T. H. Similarity of reverse transcriptase-like sequences of viruses, transposable elements, and mitochondrial introns. Mol Biol Evol. 1988 Nov;5(6):675–690. doi: 10.1093/oxfordjournals.molbev.a040521. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES