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. 1998 Feb 15;26(4):925–931. doi: 10.1093/nar/26.4.925

Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus.

S A Akbar Behjatnia 1, I B Dry 1, M Ali Rezaian 1
PMCID: PMC147362  PMID: 9461449

Abstract

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.

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Selected References

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  1. Argüello-Astorga G. R., Guevara-González R. G., Herrera-Estrella L. R., Rivera-Bustamante R. F. Geminivirus replication origins have a group-specific organization of iterative elements: a model for replication. Virology. 1994 Aug 15;203(1):90–100. doi: 10.1006/viro.1994.1458. [DOI] [PubMed] [Google Scholar]
  2. Baas P. D., Jansz H. S. Single-stranded DNA phage origins. Curr Top Microbiol Immunol. 1988;136:31–70. doi: 10.1007/978-3-642-73115-0_3. [DOI] [PubMed] [Google Scholar]
  3. Choi I. R., Stenger D. C. Strain-specific determinants of beet curly top geminivirus DNA replication. Virology. 1995 Feb 1;206(2):904–912. doi: 10.1006/viro.1995.1013. [DOI] [PubMed] [Google Scholar]
  4. Choi I. R., Stenger D. C. The strain-specific cis-acting element of beet curly top geminivirus DNA replication maps to the directly repeated motif of the ori. Virology. 1996 Dec 1;226(1):122–126. doi: 10.1006/viro.1996.0634. [DOI] [PubMed] [Google Scholar]
  5. Dry I. B., Krake L. R., Rigden J. E., Rezaian M. A. A novel subviral agent associated with a geminivirus: the first report of a DNA satellite. Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):7088–7093. doi: 10.1073/pnas.94.13.7088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dry I. B., Rigden J. E., Krake L. R., Mullineaux P. M., Rezaian M. A. Nucleotide sequence and genome organization of tomato leaf curl geminivirus. J Gen Virol. 1993 Jan;74(Pt 1):147–151. doi: 10.1099/0022-1317-74-1-147. [DOI] [PubMed] [Google Scholar]
  7. Eagle P. A., Orozco B. M., Hanley-Bowdoin L. A DNA sequence required for geminivirus replication also mediates transcriptional regulation. Plant Cell. 1994 Aug;6(8):1157–1170. doi: 10.1105/tpc.6.8.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fontes E. P., Eagle P. A., Sipe P. S., Luckow V. A., Hanley-Bowdoin L. Interaction between a geminivirus replication protein and origin DNA is essential for viral replication. J Biol Chem. 1994 Mar 18;269(11):8459–8465. [PubMed] [Google Scholar]
  9. Fontes E. P., Gladfelter H. J., Schaffer R. L., Petty I. T., Hanley-Bowdoin L. Geminivirus replication origins have a modular organization. Plant Cell. 1994 Mar;6(3):405–416. doi: 10.1105/tpc.6.3.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fontes E. P., Luckow V. A., Hanley-Bowdoin L. A geminivirus replication protein is a sequence-specific DNA binding protein. Plant Cell. 1992 May;4(5):597–608. doi: 10.1105/tpc.4.5.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Graves B. J., Johnson P. F., McKnight S. L. Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. Cell. 1986 Feb 28;44(4):565–576. doi: 10.1016/0092-8674(86)90266-7. [DOI] [PubMed] [Google Scholar]
  12. Gruss A., Ehrlich S. D. The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev. 1989 Jun;53(2):231–241. doi: 10.1128/mr.53.2.231-241.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haley A., Zhan X., Richardson K., Head K., Morris B. Regulation of the activities of African cassava mosaic virus promoters by the AC1, AC2, and AC3 gene products. Virology. 1992 Jun;188(2):905–909. doi: 10.1016/0042-6822(92)90551-y. [DOI] [PubMed] [Google Scholar]
  14. Heyraud-Nitschke F., Schumacher S., Laufs J., Schaefer S., Schell J., Gronenborn B. Determination of the origin cleavage and joining domain of geminivirus Rep proteins. Nucleic Acids Res. 1995 Mar 25;23(6):910–916. doi: 10.1093/nar/23.6.910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ilyina T. V., Koonin E. V. Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res. 1992 Jul 11;20(13):3279–3285. doi: 10.1093/nar/20.13.3279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jupin I., Hericourt F., Benz B., Gronenborn B. DNA replication specificity of TYLCV geminivirus is mediated by the amino-terminal 116 amino acids of the Rep protein. FEBS Lett. 1995 Apr 3;362(2):116–120. doi: 10.1016/0014-5793(95)00221-t. [DOI] [PubMed] [Google Scholar]
  17. Koonin E. V., Ilyina T. V. Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins. J Gen Virol. 1992 Oct;73(Pt 10):2763–2766. doi: 10.1099/0022-1317-73-10-2763. [DOI] [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. Laufs J., Traut W., Heyraud F., Matzeit V., Rogers S. G., Schell J., Gronenborn B. In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3879–3883. doi: 10.1073/pnas.92.9.3879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lazarowitz S. G., Wu L. C., Rogers S. G., Elmer J. S. Sequence-specific interaction with the viral AL1 protein identifies a geminivirus DNA replication origin. Plant Cell. 1992 Jul;4(7):799–809. doi: 10.1105/tpc.4.7.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Noris E., Jupin I., Accotto G. P., Gronenborn B. DNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus. Virology. 1996 Mar 15;217(2):607–612. doi: 10.1006/viro.1996.0157. [DOI] [PubMed] [Google Scholar]
  22. Orozco B. M., Miller A. B., Settlage S. B., Hanley-Bowdoin L. Functional domains of a geminivirus replication protein. J Biol Chem. 1997 Apr 11;272(15):9840–9846. doi: 10.1074/jbc.272.15.9840. [DOI] [PubMed] [Google Scholar]
  23. Papavassiliou A. G. 1,10-Phenanthroline-copper ion nuclease footprinting of DNA-protein complexes in situ following mobility-shift electrophoresis assays. Methods Mol Biol. 1994;30:43–78. doi: 10.1385/0-89603-256-6:43. [DOI] [PubMed] [Google Scholar]
  24. Ragnhildstveit E., Fjose A., Becker P. B., Quivy J. P. Solid phase technology improves coupled gel shift/footprinting analysis. Nucleic Acids Res. 1997 Jan 15;25(2):453–454. doi: 10.1093/nar/25.2.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Revington G. N., Sunter G., Bisaro D. M. DNA sequences essential for replication of the B genome component of tomato golden mosaic virus. Plant Cell. 1989 Oct;1(10):985–992. doi: 10.1105/tpc.1.10.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Saunders K., Lucy A., Stanley J. DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication. Nucleic Acids Res. 1991 May 11;19(9):2325–2330. doi: 10.1093/nar/19.9.2325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Saunders K., Lucy A., Stanley J. RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus. Nucleic Acids Res. 1992 Dec 11;20(23):6311–6315. doi: 10.1093/nar/20.23.6311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stanley J. Analysis of African cassava mosaic virus recombinants suggests strand nicking occurs within the conserved nonanucleotide motif during the initiation of rolling circle DNA replication. Virology. 1995 Jan 10;206(1):707–712. doi: 10.1016/s0042-6822(95)80093-x. [DOI] [PubMed] [Google Scholar]
  30. Stenger D. C., Revington G. N., Stevenson M. C., Bisaro D. M. Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8029–8033. doi: 10.1073/pnas.88.18.8029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sung Y. K., Coutts R. H. Potato yellow mosaic geminivirus AC2 protein is a sequence non-specific DNA binding protein. FEBS Lett. 1996 Mar 25;383(1-2):51–54. doi: 10.1016/0014-5793(96)00217-7. [DOI] [PubMed] [Google Scholar]
  32. Sunter G., Hartitz M. D., Bisaro D. M. Tomato golden mosaic virus leftward gene expression: autoregulation of geminivirus replication protein. Virology. 1993 Jul;195(1):275–280. doi: 10.1006/viro.1993.1374. [DOI] [PubMed] [Google Scholar]
  33. Thömmes P. A., Buck K. W. Synthesis of the tomato golden mosaic virus AL1, AL2, AL3 and AL4 proteins in vitro. J Gen Virol. 1994 Aug;75(Pt 8):1827–1834. doi: 10.1099/0022-1317-75-8-1827. [DOI] [PubMed] [Google Scholar]
  34. Thömmes P., Osman T. A., Hayes R. J., Buck K. W. TGMV replication protein AL1 preferentially binds to single-stranded DNA from the common region. FEBS Lett. 1993 Mar 15;319(1-2):95–99. doi: 10.1016/0014-5793(93)80044-u. [DOI] [PubMed] [Google Scholar]

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