, Volume 217, Issue 3, pp 457–465 | Cite as

Plant-specific regulation of replication protein A2 (OsRPA2) from rice during the cell cycle and in response to ultraviolet light exposure

  • Tanja Marwedel
  • Toyotaka Ishibashi
  • René Lorbiecke
  • Silke Jacob
  • Kengo Sakaguchi
  • Margret SauterEmail author
Original Article


DNA replication is a process that is highly conserved among eukaryotes. Nonetheless, little is known about the proteins involved in it in plants. Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein with several functions in DNA metabolism in humans and yeast and supposedly also in plants. Here we report on the regulation of OsRPA2, the 32-kDa subunit of RPA from rice (Oryza sativa L.). We found conserved regulation mechanisms at the level of gene expression between animal and plant RPA2 genes and distinct features of OsRPA2 regulation at the level of protein expression. Unlike in animals or in yeast, protein abundance in rice was regulated in a cell cycle phase-specific manner and was altered after UV-C light exposure. On the other hand, posttranslational modification through phosphorylation did not appear to play a pivotal role in rice as it does in animal cells. Our results indicate that plant-specific mechanisms of regulation have evolved for RPA2 within the generally well-conserved process of DNA replication, suggesting specific requirements for regulation of DNA metabolism in plants as compared to other eukaryotes.


Cell division cycle Growth Oryza Replication protein A S phase 



replication protein A


uracil-DNA glycosylase


xeroderma pigmentosum A



We thank Prof. A.-M. Lambert and Dr. A.-C. Schmit (Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, Strasbourg, France) for providing the anti γ-tubulin antibody.


  1. Birell GW, Giaever G, Chu AM, Davis RW, Brown M (2001) A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity. Proc Natl Acad Sci 98:12608–12613PubMedGoogle Scholar
  2. Biswas GCG, Iglesias VA, Datta SK, Potrykus I (1994) Transgenic Indica rice (Oryza sativa L.) plants obtained by direct gene transfer to protoplasts. J Biotechnol 32:1-10CrossRefPubMedGoogle Scholar
  3. Bochkareva E, Frappier L, Edwards MA, Bochkarev A (1998) The RPA2 subunit of human replication protein A contains a single-stranded DNA-binding domain. J Biol Chem 273:3932–3936CrossRefPubMedGoogle Scholar
  4. Bochkarev A, Bochkareva E, Frappier L, Edwards MA (1999) The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding. EMBO J 18:4498–4504CrossRefPubMedGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Brill SJ, Stillman B (1991) Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev 5:1589–1600PubMedGoogle Scholar
  7. Brush GS, Morrow DM, Hieter P, Kelly TJ (1996) The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast. Proc Natl Acad Sci 93:15075–15080CrossRefPubMedGoogle Scholar
  8. de Veylder L, Beeckmann T, Beemster GTS, de Almeida Engler J, Ormenese S, Maes S, Naudts M, van der Schueren E, Jacqmard A, Engler G, Inzé D (2002) Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-Dpa transcription factor. EMBO J 21:1360–1368PubMedGoogle Scholar
  9. Din S, Brill SJ, Fairman MP, Stillman B (1990) Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev 4: 968–977PubMedGoogle Scholar
  10. Erhardt M, Stoppin-Mellet V, Campagne S, Canaday J, Mutterer J, Fabian T, Sauter M, Muller T, Peter C, Lambert A-M, Schmit A-C (2002) The plant Spc98p homologue colocalizes with γ-tubulin at microtubule nucleation sites and is required for microtubule nucleation. J Cell Sci 115:2423–2431PubMedGoogle Scholar
  11. Fabian-Marwedel T, Umeda M, Sauter M (2002) The rice cyclin-dependent kinase-activating kinase R2 regulates S-phase progression. Plant Cell 14:197–210CrossRefPubMedGoogle Scholar
  12. Fang F, Newport JW (1993) Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle. J Cell Sci 106:983–994PubMedGoogle Scholar
  13. Garcia-Maya MM, Buck KW (1997) Isolation and characterization of replication protein A (RPA) from tobacco cells. FEBS 413:181–184CrossRefGoogle Scholar
  14. Ishibashi T, Kimura S, Furukawa T, Hatanaka M, Hashimoto J, Sakaguchi K (2001) Two types of replication protein A 70 kDa subunit in rice, Oryza sativa: molecular cloning, characterization and cellular and tissue distribution. Gene 272:335–343CrossRefPubMedGoogle Scholar
  15. Kenny MK, Schlegel U, Furneaux H, Hurwitz J (1990) The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J Biol Chem 265:7693–7700PubMedGoogle Scholar
  16. Kalma Y, Marash L, Lamed Y, Ginberg D (2001) Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2. Oncogene 20:1379–1387CrossRefPubMedGoogle Scholar
  17. Kosugi S, Ohashi Y (2002) E2F sites that can interact with E2F proteins cloned from rice are required for meristematic tissue-specific expression of rice and tobacco proliferating cell nuclear antigen promoters. Plant J 29:45–59CrossRefPubMedGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of maize bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  19. Lee SH, Kim DK, Drissi R (1995) Human xeroderma pigmentosum group A protein interacts with human replication protein A and inhibits DNA replication. J Biol Chem 270:21800–21805CrossRefPubMedGoogle Scholar
  20. Levenson V, Hamlin JL (1993) A general protocol for evaluating the specific effects of DNA replication inhibitors. Nucleic Acids Res 21:3997-4004PubMedGoogle Scholar
  21. Lorbiecke R, Sauter M (1998) Induction of cell growth and cell division in the intercalary meristem of submerged deepwater rice (Oryza sativa L.). Planta 204:140–145Google Scholar
  22. Lorbiecke R, Sauter M (2002) Comparative analysis of PSK peptide growth factor precurser homologs. Plant Sci 163:321–332CrossRefGoogle Scholar
  23. Magyar Z, Bakó L, Bögre L, Dedeoglu D, Kapros T, Dudits D (1993) Active cdc2 genes and cell cycle phase-specific cdc2-related kinase complexes in hormone-stimulated alfalfa cells. Plant J 4:151–161CrossRefGoogle Scholar
  24. Magyar Z, Atanasova A, De Veylder L, Rombauts S, Inzé D (2000) Characterization of two distinct DP-related genes from Arabidopsis. FEBS Lett 486:79–87PubMedGoogle Scholar
  25. Mironov V, De Veylder L, Van Montagu M, Inzé D (1999) Cyclin-dependent kinases and cell division in plants—the nexus. Plant Cell 11:509–521PubMedGoogle Scholar
  26. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497Google Scholar
  27. Murzin AG (1993) OB (oligonucleotide/oligosaccharide)-fold: common structural and functional solution for non-homologous sequences. EMBO J 12:861–867PubMedGoogle Scholar
  28. Nagelhus TA, Haug T, Singh KK, Keshav KF, Skorpen F, Otterlei M, Bharati S, Lindmo T, Benichou S, Benarous R, Krokan HE (1997) A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A. J Biol Chem 272:6561–6566CrossRefPubMedGoogle Scholar
  29. Oakley GG, Loberg LI, Yao J, Risinger MA, Yunker MF, Carty MP, Dixon K (2001) UV-induced hyperphosphorylation of replication protein A depends on DNA replication and expression of ATM protein. Mol Biol Cell 12:1199–1213PubMedGoogle Scholar
  30. Philipova D, Mullen JR, Maniar HS, Lu J, Gu C, Brill SJ (1996) A hierarchy of SSB promoters in replication protein A. Genes Dev 10:2222–2233PubMedGoogle Scholar
  31. Planchais S, Glab N, Inzé D, Bergounioux C (2000) Chemical inhibitors: a tool for plant cell cycle studies. FEBS Lett 476:78–83CrossRefPubMedGoogle Scholar
  32. Ramirez-Parra E, Xie Q, Boniotti MB, Gutierrez C (1999) The cloning of plant E2F, a retinoblastoma-binding protein, reveals unique and conserved features with animal G1/S regulators. Nucleic Acids Res 27:3527–3533PubMedGoogle Scholar
  33. Rossi V, Varotto S (2002) Insights into the G1/S transition in plants. Planta 215:345–356CrossRefPubMedGoogle Scholar
  34. Rost B, Sander C, Schneider R (1994) PHD—an automatic mail server for protein secondary structure prediction. CABIOS 10:53–60. Scholar
  35. Sauter M (1997) Differential expression of a CAK (cdc2-activating kinase)-like protein kinase, cyclins and cdc2 genes from rice during the cell cycle and in response to gibberellin. Plant J 11:181–190Google Scholar
  36. Sauter M (2000) Rice in deep water: 'How to take heed against a sea of troubles'. Naturwissenschaften 87:289–303CrossRefPubMedGoogle Scholar
  37. Sauter M, Mekhedov SL, Kende H (1995) Gibberellin promotes histone H1 kinase activity and the expression of cdc2 and cyclin genes during the induction of rapid growth in deepwater rice internodes. Plant J 7:623–632PubMedGoogle Scholar
  38. Sekine M, Ito M, Uemukai K, Maeda Y, Nakagami H, Shinmyo A (1999) Isolation and characterization of the E2F-like gene in plant. FEBS Lett 460:117–122PubMedGoogle Scholar
  39. Singh KK, Samson L (1995) Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes. Proc Natl Acad Sci 92:4907–4911PubMedGoogle Scholar
  40. Stals H, Inzé D (2001) When plant cells decide to divide. Trends Plant Sci 6:359–364PubMedGoogle Scholar
  41. Treuner K, Ramsperger U, Knippers R (1996) Replication protein A induces the unwinding of long double-stranded DNA regions. J Mol Biol 259:104–112CrossRefPubMedGoogle Scholar
  42. Treuner K, Findeisen M, Strausfeld U, Knippers R (1999a) Phosphorylation of replication protein A middle subunit (RPA2) leads to disassembly of the RPA heterotrimer. J Biol Chem 274:15556–15561CrossRefPubMedGoogle Scholar
  43. Treuner K, Okuyama A, Knippers R, Fackelmayer FO (1999b) Hyperphosphorylation of replication protein A middle subunit (RPA2) in apoptosis. Nucleic Acids Res 27:1499–1504CrossRefPubMedGoogle Scholar
  44. van der Knaap E, Jagoueix S, Kende H (1997) Expression of an ortholog of replication protein A1 (RPA1) is induced by gibberellin in deepwater rice. Proc Natl Acad Sci 94:9979–9983CrossRefPubMedGoogle Scholar
  45. Wold MS (1997) Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu Rev Biochem 66:61–92PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Tanja Marwedel
    • 1
  • Toyotaka Ishibashi
    • 2
  • René Lorbiecke
    • 1
  • Silke Jacob
    • 1
  • Kengo Sakaguchi
    • 2
  • Margret Sauter
    • 1
    Email author
  1. 1.Institut für Allgemeine BotanikUniversität HamburgHamburgGermany
  2. 2.Department of Applied Biological Science, Faculty of Science and TechnologyScience University of TokyoNoda-shi, Chiba-ken, TokyoJapan

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