Abstract
The Rep protein of geminiviruses is the sole viral protein required for their DNA replication. The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop). Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro. Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity. In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells. These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication.
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- Berchtold H., Reshetnikova L., Reiser C. O., Schirmer N. K., Sprinzl M., Hilgenfeld R. Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature. 1993 Sep 9;365(6442):126–132. doi: 10.1038/365126a0. [DOI] [PubMed] [Google Scholar]
- Chejanovsky N., Carter B. J. Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant negative phenotype for DNA replication. J Virol. 1990 Apr;64(4):1764–1770. doi: 10.1128/jvi.64.4.1764-1770.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clark R., Lane D. P., Tjian R. Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity. J Biol Chem. 1981 Nov 25;256(22):11854–11858. [PubMed] [Google Scholar]
- Cox M. M. Why does RecA protein hydrolyse ATP? Trends Biochem Sci. 1994 May;19(5):217–222. doi: 10.1016/0968-0004(94)90025-6. [DOI] [PubMed] [Google Scholar]
- 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]
- Fütterer J., Gordon K., Pfeiffer P., Sanfaçon H., Pisan B., Bonneville J. M., Hohn T. Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader. Virus Genes. 1989 Sep;3(1):45–55. doi: 10.1007/BF00301986. [DOI] [PubMed] [Google Scholar]
- Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gorbalenya A. E., Koonin E. V., Wolf Y. I. A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses. FEBS Lett. 1990 Mar 12;262(1):145–148. doi: 10.1016/0014-5793(90)80175-i. [DOI] [PubMed] [Google Scholar]
- 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]
- Heyraud F., Matzeit V., Kammann M., Schaefer S., Schell J., Gronenborn B. Identification of the initiation sequence for viral-strand DNA synthesis of wheat dwarf virus. EMBO J. 1993 Nov;12(11):4445–4452. doi: 10.1002/j.1460-2075.1993.tb06130.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heyraud F., Matzeit V., Schaefer S., Schell J., Gronenborn B. The conserved nonanucleotide motif of the geminivirus stem-loop sequence promotes replicational release of virus molecules from redundant copies. Biochimie. 1993;75(7):605–615. doi: 10.1016/0300-9084(93)90067-3. [DOI] [PubMed] [Google Scholar]
- Hofer J. M., Dekker E. L., Reynolds H. V., Woolston C. J., Cox B. S., Mullineaux P. M. Coordinate regulation of replication and virion sense gene expression in wheat dwarf virus. Plant Cell. 1992 Feb;4(2):213–223. doi: 10.1105/tpc.4.2.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Honegger A. M., Dull T. J., Felder S., Van Obberghen E., Bellot F., Szapary D., Schmidt A., Ullrich A., Schlessinger J. Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell. 1987 Oct 23;51(2):199–209. doi: 10.1016/0092-8674(87)90147-4. [DOI] [PubMed] [Google Scholar]
- 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]
- John J., Sohmen R., Feuerstein J., Linke R., Wittinghofer A., Goody R. S. Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. Biochemistry. 1990 Jun 26;29(25):6058–6065. doi: 10.1021/bi00477a025. [DOI] [PubMed] [Google Scholar]
- Jurnak F. Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science. 1985 Oct 4;230(4721):32–36. doi: 10.1126/science.3898365. [DOI] [PubMed] [Google Scholar]
- Kheyr-Pour A., Bendahmane M., Matzeit V., Accotto G. P., Crespi S., Gronenborn B. Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus. Nucleic Acids Res. 1991 Dec 25;19(24):6763–6769. doi: 10.1093/nar/19.24.6763. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Lessl M., Lanka E. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell. 1994 May 6;77(3):321–324. doi: 10.1016/0092-8674(94)90146-5. [DOI] [PubMed] [Google Scholar]
- Lin J. J., Phillips A. M., Hearst J. E., Sancar A. Active site of (A)BC excinuclease. II. Binding, bending, and catalysis mutants of UvrB reveal a direct role in 3' and an indirect role in 5' incision. J Biol Chem. 1992 Sep 5;267(25):17693–17700. [PubMed] [Google Scholar]
- Matzeit V., Schaefer S., Kammann M., Schalk H. J., Schell J., Gronenborn B. Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants. Plant Cell. 1991 Mar;3(3):247–258. doi: 10.1105/tpc.3.3.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Navot N., Pichersky E., Zeidan M., Zamir D., Czosnek H. Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component. Virology. 1991 Nov;185(1):151–161. doi: 10.1016/0042-6822(91)90763-2. [DOI] [PubMed] [Google Scholar]
- Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pomerantz J. L., Sharp P. A., Pabo C. O. Structure-based design of transcription factors. Science. 1995 Jan 6;267(5194):93–96. doi: 10.1126/science.7809612. [DOI] [PubMed] [Google Scholar]
- Rehrauer W. M., Kowalczykowski S. C. Alteration of the nucleoside triphosphate (NTP) catalytic domain within Escherichia coli recA protein attenuates NTP hydrolysis but not joint molecule formation. J Biol Chem. 1993 Jan 15;268(2):1292–1297. [PubMed] [Google Scholar]
- 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]
- Schlichting I., Almo S. C., Rapp G., Wilson K., Petratos K., Lentfer A., Wittinghofer A., Kabsch W., Pai E. F., Petsko G. A. Time-resolved X-ray crystallographic study of the conformational change in Ha-Ras p21 protein on GTP hydrolysis. Nature. 1990 May 24;345(6273):309–315. doi: 10.1038/345309a0. [DOI] [PubMed] [Google Scholar]
- Seeley T. W., Grossman L. The role of Escherichia coli UvrB in nucleotide excision repair. J Biol Chem. 1990 May 5;265(13):7158–7165. [PubMed] [Google Scholar]
- Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
- 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]
- Story R. M., Steitz T. A. Structure of the recA protein-ADP complex. Nature. 1992 Jan 23;355(6358):374–376. doi: 10.1038/355374a0. [DOI] [PubMed] [Google Scholar]
- Sung P., Higgins D., Prakash L., Prakash S. Mutation of lysine-48 to arginine in the yeast RAD3 protein abolishes its ATPase and DNA helicase activities but not the ability to bind ATP. EMBO J. 1988 Oct;7(10):3263–3269. doi: 10.1002/j.1460-2075.1988.tb03193.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- la Cour T. F., Nyborg J., Thirup S., Clark B. F. Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography. EMBO J. 1985 Sep;4(9):2385–2388. doi: 10.1002/j.1460-2075.1985.tb03943.x. [DOI] [PMC free article] [PubMed] [Google Scholar]