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An Arabidopsis homologue of bacterial RecA that complements an E. coli recA deletion is targeted to plant mitochondria

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Abstract

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mtrecA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mtrecA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mtrecA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mtrecA in homologous recombination and/or repair in Arabidopsis mitochondria.

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References

  • Akashi K, Grandjean O, Small I (1998) Potential dual targeting of an Arabidopsis archaebacterial-like histidyl-tRNA synthetase to mitochondria and chloroplasts. FEBS Lett 431:39–44

    Article  CAS  PubMed  Google Scholar 

  • Andersson SG, Zomorodipour A, Andersson JO, Sicheritz-Ponten T, Alsmark UC, Podowski RM, Naslund AK, Eriksson AS, Winkler HH, Kurland CG (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396:133–140

    CAS  PubMed  Google Scholar 

  • Angulo JF, Schwencke J, Moreau PL, Moustacchi E, Devoret R (1985) A yeast protein analogous to Escherichia coli RecA protein whose cellular level is enhanced after UV irradiation. Mol Gen Genet 201:20–24

    CAS  PubMed  Google Scholar 

  • Cao J, Combs C, Jagendorf AT (1997) The chloroplast-located homolog of bacterial DNA recombinase. Plant Cell Physiol 38:1319–1325

    CAS  PubMed  Google Scholar 

  • Cerutti H, Osman M, Grandoni P, Jagendorf AT (1992) A homolog of Escherichia coli RecA protein in plastids of higher plants. Proc Natl Acad Sci USA 89:8068–8072

    CAS  PubMed  Google Scholar 

  • Chow KS, Singh DP, Roper JM, Smith AG (1997) A single precursor protein for ferrochelatase-I from Arabidopsis is imported in vitro into both chloroplasts and mitochondria. J Biol Chem 272:27565–27561

    Article  CAS  PubMed  Google Scholar 

  • Conley CA, Hanson MR (1995) How do alterations in plant mitochondrial genomes disrupt pollen development? J Bioenerg Biomembr 27:447–457

    Google Scholar 

  • Cox MM (2000) Recombinational DNA repair in bacteria and the RecA protein. Prog Nucleic Acid Res Mol Biol 63:311–366

    CAS  Google Scholar 

  • Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984

    PubMed  Google Scholar 

  • Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016

    Article  CAS  PubMed  Google Scholar 

  • Foury F, Lahaye A (1987) Cloning and sequencing of the PIF gene involved in repair and recombination of yeast mitochondrial DNA. EMBO J 6:1441–1449

    CAS  PubMed  Google Scholar 

  • Frei C, Gasser SM (2000) RecQ helicases: the DNA replication checkpoint connection. J Cell Sci 113:2641–2646

    CAS  PubMed  Google Scholar 

  • Geiss KT, Abbas GM, Makaroff CA (1994) Intron loss from the NADH dehydrogenase subunit 4 gene of lettuce mitochondrial DNA: evidence for homologous recombination of a cDNA intermediate. Mol Gen Genet 243:97–105

    CAS  PubMed  Google Scholar 

  • Gutierres S, Combettes B, De Paepe R, Mirande M, Lelandais C, Vedel F, Chetrit P (1999) In the Nicotiana sylvestris CMSII mutant, a recombination-mediated change 5´ to the first exon of the mitochondrial nad1 gene is associated with lack of the NADH:ubiquinone oxidoreductase (complex I) NAD1 subunit. Eur J Biochem 261:361–370

    Article  CAS  PubMed  Google Scholar 

  • Harmon FG, Kowalczykowski SC (1998) RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination. Genes Dev 12:1134–1144

    CAS  PubMed  Google Scholar 

  • Hartung F, Plchova H, Puchta H (2000) Molecular characterization of RecQ homologues in Arabidopsis thaliana. Nucleic Acids Res 28:4275–4282

    Article  CAS  PubMed  Google Scholar 

  • Hedtke B, Legen J, Weihe A, Hermann RG, Börner T (2002) Six active phage-type RNA polymerase genes in Nicotiana tabacum. Plant J 30:625–637

    Article  PubMed  Google Scholar 

  • Hong EL, Shinohara A, Bishop DK (2001) Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single- strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA. J Biol Chem 276:41906–41912

    Article  CAS  PubMed  Google Scholar 

  • Hrubec T, Robinson J, Donaldson R (1985) Isolation of mitochondria from soybean leaves on discontinuous Percoll gradients. Plant Physiol 77:1010–1012

    CAS  Google Scholar 

  • Kajander OA, Karhunen PJ, Holt IJ, Jacobs HT (2001) Prominent mitochondrial DNA recombination intermediates in human heart muscle. EMBO Rep 2:1007–1012

    CAS  PubMed  Google Scholar 

  • Klein M, Eckert-Ossenkopp U, Schmiedeberg I, Brandt P, Unseld M, Brennicke A, Schuster W (1994) Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones. Plant J 6:447–455

    Article  CAS  PubMed  Google Scholar 

  • Kobayashi Y, Dokiya Y, Sugita M (2001) Dual targeting of phage-type RNA polymerase to both mitochondria and plastids is due to alternative translation initiation in single transcripts. Biochem Biophys Res Comm 289:1106–1113

    Article  CAS  PubMed  Google Scholar 

  • Kowalczykowski SC (2000) Initiation of genetic recombination and recombination-dependent replication. Trends Biochem Sci 25:156–165

    Article  CAS  PubMed  Google Scholar 

  • Larsson NG, Clayton DA (1995) Molecular genetic aspects of human mitochondrial disorders. Ann Rev Genet 29:151–178

    Article  CAS  PubMed  Google Scholar 

  • Ling F, Makishima F, Morishima N, Shibata T (1995) A nuclear mutation defective in mitochondrial recombination in yeast. EMBO J 14:4090–4101

    Google Scholar 

  • Lockshon D, Zweifel SG, Freeman-Cook LL, Lorimer HE, Brewer BJ, Fangman WL (1995) A role for recombination junctions in the segregation of mitochondrial DNA in yeast. Cell 81:947–955

    CAS  PubMed  Google Scholar 

  • MacAlpine DM, Perlman PS, Butow RA (1998) The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo. Proc Natl Acad Sci USA 95:6739–6743

    CAS  PubMed  Google Scholar 

  • Manna F, Massardo DR, Del Giudice L, Buonocore A, Nappo AG, Alifano P, Schafer B, Wolf K (1991) The mitochondrial genome of Schizosaccharomyces pombe. Stimulation of intra-chromosomal recombination in Escherichia coli by the gene product of the first cox1 intron. Curr Genet 19:295–299

    CAS  PubMed  Google Scholar 

  • Marienfeld J, Unseld M, Brandt P, Brennicke A (1996) Genomic recombination of the mitochondrial atp6 gene in Arabidopsis thaliana at the protein processing site creates two different presequences. DNA Res 3:287–290

    CAS  PubMed  Google Scholar 

  • Marienfeld JR, Unseld M, Brandt P, Brennicke A (1997) Mosaic open reading frames in the Arabidopsis thaliana mitochondrial genome. Biol Chem 378:859–862

    CAS  PubMed  Google Scholar 

  • Muise RC, Hauswirth WW (1995) Selective DNA amplification regulates transcript levels in plant mitochondria. Curr Genet 28:113–121

    CAS  PubMed  Google Scholar 

  • Nakai K (2000) Protein sorting signals and prediction of subcellular localization. Adv Protein Chem 54:277–344

    CAS  PubMed  Google Scholar 

  • Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–36

    CAS  PubMed  Google Scholar 

  • Ogawa T, Shinohara A, Nabetani A, Ikeya T, Yu X, Egelman EH, Ogawa H (1993) RecA-like recombination proteins in eukaryotes: functions and structures of RAD51 genes. Cold Spring Harb Symp Quant Biol 58:567–576

    CAS  PubMed  Google Scholar 

  • Pang Q, Hays JB, Rajagopal I (1992) A plant cDNA that partially complements Escherichia coli rec A mutations predicts a polypeptide not strongly homologous to RecA proteins. Proc Natl Acad Sci USA 89:8073–8077

    CAS  PubMed  Google Scholar 

  • Perry SE, Li HM, Keegstra K (1991) In vitro reconstitution of protein transport into chloroplasts. Methods Cell Biol 34:327–343

    CAS  PubMed  Google Scholar 

  • Sato S, Hotta Y, Tabata S (1995) Structural analysis of a recA -like gene in the genome of Arabidopsis thaliana. DNA Res 2:89–93

    CAS  PubMed  Google Scholar 

  • Scharfe C, Zaccaria P, Hoertnagel K, Jaksch J, Klopstock T, Lill R, Prokisch H, Gerbitz KD, Mewes HW, Meitinger T (1999) MITOP, database for mitochondria-related proteins, genes and diseases. Nucleic Acids Res 27:153–155

    Article  CAS  PubMed  Google Scholar 

  • Scharfe C, Zaccaria P, Hoertnagel K, Jaksch J, Klopstock T, Dembowski M, Lill R, Prokisch H, Gerbitz KD, Neupert W, Mewes HW, Meitinger T (2000) MITOP, the mitochondrial proteome database: 2000 update. Nucleic Acids Res 28:155–158

    Article  CAS  PubMed  Google Scholar 

  • Sena EP, Revet B, Moustacchi E (1986) In vivo homologous recombination intermediates of yeast mitochondrial DNA analyzed by electron microscopy. Mol Gen Genet 202:421–428

    CAS  PubMed  Google Scholar 

  • Silva-Filho MD, Wieers MC, Flugge UI, Chaumont F, Boutry M (1997) Different in vitro and in vivo targeting properties of the transit peptide of a chloroplast envelope inner membrane protein. J Biol Chem 272:15264–15269

    Article  CAS  PubMed  Google Scholar 

  • Small I, Wintz H, Akashi K, Mireau H (1998) Two birds with one stone: genes that encode products targeted to two or more compartments. Plant Mol Biol 38:265–277

    Article  CAS  PubMed  Google Scholar 

  • Small WC, McAlister-Henn L (1997) Metabolic effects of altering redundant targeting signals for yeast mitochondrial malate dehydrogenase. Arch Biochem Biophys 344:53–60

    Article  CAS  PubMed  Google Scholar 

  • Thyagarajan B, Padua RA, Campbell C (1996) Mammalian mitochondria possess homologous DNA recombination activity. J Biol Chem 271:27536–27543

    CAS  PubMed  Google Scholar 

  • Unseld M, Marienfeld JR, Brandt P, Brennicke A (1997) The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nat Genet 15:57–61

    CAS  PubMed  Google Scholar 

  • Watanabe N, Che FS, Iwano M, Takayama S, Yoshida S, Isogai A (2001) Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame initiation codons. J Biol Chem 276:20474–20481

    Article  CAS  PubMed  Google Scholar 

  • Weigel D, Glazebrook J (2002) Arabidopsis: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

  • Wu L, Hickson ID (2001) RecQ helicases and topoisomerases: components of a conserved complex for the regulation of genetic recombination. Cell Mol Life Sci 58:894–901

    CAS  PubMed  Google Scholar 

  • Yakhnin AV, Vinokurov LM, Surin AK, Alakhov YB (1998) Green fluorescent protein purification by organic extraction. Protein Expr Purif 14:382–386

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Drs. Narendra Singh, William McCleary and Allan Caplan for helpful discussions, Dr. Andre T. Jagendorf for the polyclonal antibodies against chloroplast RecA and Dr. Tom Elthon for monoclonal antibodies against mitochondrial porin. We also thank Dr. David McClellan for assistance with the phylogenetic analyses, and Paul Gunn and Sean Echols for help with various aspects of the work. We acknowledge support from the NIH, the COSAM Dean's Research Initiative (Auburn University), the Alabama Agricultural Experiment Station, and the Mentoring Environment Grant Program of the BYU Office of Research and Creative Activities

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  1. F. R. Khazi

    Present address: Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104, USA

Authors and Affiliations

  1. Department of Biological Sciences, Auburn University, AL 36849, Auburn, USA

    F. R. Khazi

  2. Department of Microbiology and Molecular Biology, 775 WIDB, Brigham Young University, Provo, UT 84602, USA

    A. C. Edmondson & B. L. Nielsen

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  1. F. R. Khazi
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  2. A. C. Edmondson
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  3. B. L. Nielsen
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Correspondence to B. L. Nielsen.

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Communicated by R. G. Herrmann

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Khazi, F.R., Edmondson, A.C. & Nielsen, B.L. An Arabidopsis homologue of bacterial RecA that complements an E. coli recA deletion is targeted to plant mitochondria. Mol Gen Genomics 269, 454–463 (2003). https://doi.org/10.1007/s00438-003-0859-6

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