Conserved Sequence and Structural Motifs Contribute to the DNA Binding and Cleavage Activities of a Geminivirus Replication Protein
1998; Elsevier BV; Volume: 273; Issue: 38 Linguagem: Inglês
10.1074/jbc.273.38.24448
ISSN1083-351X
AutoresBeverly M. Orozco, Linda Hanley‐Bowdoin,
Tópico(s)Animal Virus Infections Studies
ResumoTomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1–120) and oligomerization (amino acids 134–181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitroexperiments demonstrated that a pair of predicted α-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions. Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1–120) and oligomerization (amino acids 134–181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitroexperiments demonstrated that a pair of predicted α-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions. tomato golden mosaic virus intergenic region rolling circle replication bean golden mosaic virus tomato yellow leaf curl virus glutathione S-transferase nucleotide(s). Tomato golden mosaic virus (TGMV)1 is a member of the geminivirus family of plant-infecting viruses characterized by twin icosahedral particles and small, single-stranded DNA genomes (reviewed in Refs. 1Timmermans M.C.P. Das O.P. Messing J. Annu. Rev. Plant Physiol. 1994; 45: 79-112Crossref Scopus (103) Google Scholar and 2Hanley-Bowdoin, L., Settlage, S. B., Orozco, B. M., Nagar, S., and Robertson, D. (1998) Crit. Rev. Plant Sci., in pressGoogle Scholar). The single-stranded DNA is converted to a double-stranded form in the nucleus of infected cells and then serves as a template for rolling circle replication (RCR; Refs. 3Saunders K. Lucy A. Stanley J. Nucleic Acids Res. 1991; 19: 2325-2330Crossref PubMed Scopus (167) Google Scholar, 4Stenger D.C. Revington G.N. Stevenson M.C. Bisaro D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8029-8033Crossref PubMed Scopus (289) Google Scholar, 5Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (90) Google Scholar) and viral gene transcription (6Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar, 7Sunter G. Bisaro D.M. Virology. 1989; 173: 647-655Crossref PubMed Scopus (47) Google Scholar). Geminiviruses encode only a few proteins for these processes and depend on host DNA and RNA polymerases as well as their accessory factors. These characteristics make geminiviruses excellent model systems for studying plant DNA replication and transcription mechanisms. The TGMV genome consists of two circular DNA molecules, designated as A and B (8Hamilton W.D.O. Bisaro D.M. Coutts R.H.A. Buck K.W. Nucleic Acids Res. 1983; 11: 7387-7396Crossref PubMed Scopus (92) Google Scholar). Both components have a conserved 5′ intergenic region (IR) that separates divergent open reading frames (9Bisaro D.M. Hamilton W.D.O. Coutts R.H.A. Buck K.W. Nucleic Acids Res. 1982; 10: 4913-4922Crossref PubMed Scopus (52) Google Scholar). The IR includes the plus-strand origin of replication (10Orozco B.M. Gladfelter H.J. Settlage S.B. Eagle P.A. Gentry R. Hanley-Bowdoin L. Virology. 1998; 242: 346-356Crossref PubMed Scopus (68) Google Scholar) and the promoters for leftward and rightward transcription (6Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar, 7Sunter G. Bisaro D.M. Virology. 1989; 173: 647-655Crossref PubMed Scopus (47) Google Scholar). A directly repeated sequence in the TGMV IR is required for recognition of the plus-strand origin and negative regulation of the overlapping promoter for leftward transcription (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar, 12Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar). Related motifs are found in the genomes of most dicot-infecting geminiviruses (13Arguello-Astorga G.R. Guevara-Gonzalez R.G. Herrera-Estrella L.R. Rivera-Bustamante R.F. Virology. 1994; 203: 90-100Crossref PubMed Scopus (278) Google Scholar), and their roles in virus-specific replication have been confirmed for bean golden mosaic virus (BGMV) and beet curly top virus (14Fontes E.P.B. Gladfelter H.J. Schaffer R.L. Petty I.T.D. Hanley-Bowdoin L. Plant Cell. 1994; 6: 405-416Crossref PubMed Scopus (171) Google Scholar, 15Choi I.R. Stenger D.C. Virology. 1995; 206: 904-912Crossref PubMed Scopus (64) Google Scholar, 16Gladfelter H.J. Eagle P.A. Fontes E.P.B. Batts L.A. Hanley-Bowdoin L. Virology. 1997; 239: 186-197Crossref PubMed Scopus (47) Google Scholar). The IR also contains a hairpin with a 9-base pair loop sequence conserved among all geminiviruses that is cleaved during initiation and termination of RCR (5Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (90) Google Scholar, 17Laufs J. Traut W. Heyraud F. Matzeit V. Rogers S.G. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3879-3883Crossref PubMed Scopus (260) Google Scholar, 18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). Genetic experiments established that the hairpin structure is essential for TGMV replication (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). TGMV encodes two proteins, AL1 and AL3, that are required for efficient viral replication. AL1 is necessary for replication, whereas AL3 enhances viral DNA accumulation by an unknown mechanism (19Elmer J.S. Brand L. Sunter G. Gardiner W.E. Bisaro D.M. Rogers S.G. Nucleic Acids Res. 1988; 16: 7043-7060Crossref PubMed Scopus (212) Google Scholar, 20Sunter G. Hartitz M.D. Hormuzdi S.G. Brough C.L. Bisaro D.M. Virology. 1990; 179: 69-77Crossref PubMed Scopus (167) Google Scholar). AL1 is a multifunctional protein that confers virus-specific recognition to its cognate plus-strand origin (15Choi I.R. Stenger D.C. Virology. 1995; 206: 904-912Crossref PubMed Scopus (64) Google Scholar, 16Gladfelter H.J. Eagle P.A. Fontes E.P.B. Batts L.A. Hanley-Bowdoin L. Virology. 1997; 239: 186-197Crossref PubMed Scopus (47) Google Scholar, 21Jupin I. Hericourt F. Benz B. Gronenborn B. FEBS Lett. 1995; 362: 116-120Crossref PubMed Scopus (74) Google Scholar) and initiates RCR (17Laufs J. Traut W. Heyraud F. Matzeit V. Rogers S.G. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3879-3883Crossref PubMed Scopus (260) Google Scholar, 18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar,22Heyraud-Nitschke F. Schumacher S. Laufs J. Schaefer S. Schell J. Gronenborn B. Nucleic Acids Res. 1995; 23: 910-916Crossref PubMed Scopus (143) Google Scholar). It also actively represses its own transcription in a virus-specific manner (12Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar, 16Gladfelter H.J. Eagle P.A. Fontes E.P.B. Batts L.A. Hanley-Bowdoin L. Virology. 1997; 239: 186-197Crossref PubMed Scopus (47) Google Scholar, 23Sunter G. Hartitz M.D. Bisaro D.M. Virology. 1993; 195: 275-280Crossref PubMed Scopus (100) Google Scholar, 24Eagle P.A. Hanley-Bowdoin L. J. Virol. 1997; 71: 6947-6955Crossref PubMed Google Scholar) and induces the expression of a host DNA synthesis protein, proliferating cell nuclear antigen, in nondividing plant cells (25Nagar S. Pedersen T.J. Carrick K. Hanley-Bowdoin L. Robertson D. Plant Cell. 1995; 7: 705-719Crossref PubMed Scopus (156) Google Scholar). Several biochemical activities have been described for AL1 in vitro. TGMV AL1 binds to double-stranded DNA at the conserved repeated motif in the plus-strand origin (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar, 26Fontes E.P.B. Luckow V.A. Hanley-Bowdoin L. Plant Cell. 1992; 4: 597-608Crossref PubMed Scopus (136) Google Scholar) and cleaves single-stranded DNA in the invariant sequence of the hairpin loop (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). Analysis of the C1 protein of tomato yellow leaf curl virus (TYLCV), a TGMV AL1 homologue, revealed that it covalently attaches to the 5′ end of the cleaved DNA by a phosphotyrosyl bond and catalyzes ligation of the cleavage products (27Laufs J. Schumacher S. Geisler N. Jupin I. Gronenborn B. FEBS Lett. 1995; 377: 258-262Crossref PubMed Scopus (67) Google Scholar). ATPase activity has also been demonstrated for the AL1/C1 proteins from TYLCV and TGMV (28Desbiez C. David C. Mettouchi A. Laufs J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5640-5644Crossref PubMed Scopus (106) Google Scholar, 29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). TGMV AL1 is involved in several protein interactions. It forms large multimeric complexes (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), binds AL3 (30Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar), and interacts with a maize homologue of the animal cell cycle regulatory protein, retinoblastoma (31Ach R.A. Durfee T. Miller A.B. Taranto P. Hanley-Bowdoin L. Zambriski P.C. Gruissem W. Mol. Cell. Biol. 1997; 17: 5077-5086Crossref PubMed Scopus (202) Google Scholar). The C1 protein from wheat dwarf virus also interacts with retinoblastoma proteins from human and maize (32Xie Q. Suarezlopez P. Gutierrez C. EMBO J. 1995; 14: 4073-4082Crossref PubMed Scopus (156) Google Scholar, 33Collin S. Fernandez-Lobato M. Gooding P.S. Mullineaux P.M. Fenoll C. Virology. 1996; 219: 324-329Crossref PubMed Scopus (66) Google Scholar, 34Xie Q. Sanzburgos P. Hannon G.J. Gutierrez C. EMBO J. 1996; 15: 4900-4908Crossref PubMed Scopus (195) Google Scholar). In an earlier study, we used truncated proteins produced in a baculovirus expression system to map the regions of TGMV AL1 that are responsible for DNA cleavage, DNA binding and oligomerization (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These experiments showed that the DNA cleavage domain is located in the first 120 amino acids of AL1 whereas the oligomerization domain maps to the protein center between amino acids 121 and 181. DNA binding requires a larger region that fully overlaps the DNA cleavage and oligomerization domains. Based on these experiments, we proposed that additional amino acids between position 121 and 181 are necessary for AL1/DNA binding and/or that AL1 complex formation is required for DNA binding. In this study, we identified key sequence and structural motifs in the DNA binding and cleavage domains. A series of site-directed mutations in the AL1 N terminus were analyzed for their impact on function in vivo and in vitro. These studies focused on three amino acid motifs, which are conserved among all geminivirus AL1/C1 proteins and many initiator proteins from other RCR systems (35Koonin E.V. Ilyina T.V. J. Gen. Virol. 1992; 73: 2763-2766Crossref PubMed Scopus (132) Google Scholar, 36Ilyina T.V. Koonin E.V. Nucleic Acids Res. 1992; 20: 3279-3285Crossref PubMed Scopus (508) Google Scholar), and on a predicted helix-loop-helix motif in the N termini of AL1/C1 proteins of dicot-infecting geminiviruses (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We also used a heterologous protein interaction domain to assess the importance of oligomerization for AL1/DNA binding and coupled DNA cleavage/ligation. The plasmid pNSB148, which contains the AL1 coding sequence in a pUC118 background, was used as the template for site-directed mutagenesis (37Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 482-492Google Scholar). The oligonucleotide primers and resulting clones are listed in Table I. The sequences of fragments containing the mutations and used for subsequent cloning were verified by DNA sequence analysis. Plant expression cassettes with the mutant AL1 coding sequences were generated by subcloning NdeI/SalI fragments (AL1 amino acids 1–120) from the mutant clones into the same sites in a wild type AL1 plant expression cassette pMON1549 (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). In pMON1549, AL1 expression is under the control of the cauliflower mosaic virus 35 S promoter with a duplicated enhancer (38Kay R. Chan A. Daly M. McPherson J. Science. 1987; 236: 1299-1302Crossref PubMed Scopus (736) Google Scholar) and the 3′ end from the pea E9 rbcS gene (39Coruzzi G. Broglie R. Edwards C. Chua N.-H. EMBO J. 1984; 3: 1671-1679Crossref PubMed Scopus (208) Google Scholar). Baculovirus expression vectors coding for AL1 proteins fused to a glutathione S-transferase tag (GST-AL1) were generated by digesting the mutant plant expression cassettes with NdeI and BamHI and repairing the ends with Escherichia coli DNA polymerase I (Klenow). The fragments, which included complete AL1 coding regions, were inserted into the SmaI site of pNSB314. The pNSB314 vector contains the GST coding sequence, followed by a glycine linker, a thrombin cleavage site, and a multiple cloning site for generating in-frame fusion proteins (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). The mutant GST-AL1 expression cassettes are listed in Table I.Table IAL1 mutationsMotifMutationOligonucleotideBaculovirus vectorPlant expressionHelix 1H1–1GAGAAAGTGATgCggCcgcGGACAAGGAGCACpNSB627pNSB655H1–2GTAATTGAGAAAGTacTTCTTCTTTGGACpNSB434Helix 2H2–1CTTCATGAAGCgCTgcaCAGATTgcTATGAATTTTTTGTTAATCGGpNSB628pNSB656H2–2CTCTGCAGATcTTTccGAATTTTTTGTTAATCGGpNSB629pNSB657H2–3CATCTTCATGtcGCTCTtcGCAGAcTTTTATGAATTTTTTGTTAATCGGpNSB630pNSB658Motif 1M1–1GCACTGAGGAgcgGccgcAgcATAATTTTTGGCATpNSB649pNSB652M1–2CACCTCGAGGATAcGTAAGAgcATAATTTTTGGCATTpNSB685pNSB681M1–3GACATGAGGAgcgGTAAGAAAATAATTTTTGGGGCATTpNSB686pNSB682Motif 2M2–1GAATAAGCACGgcggccgcAGGTTGCCCATCpNSB650pNSB653Motif 3M3–1GAGTATCTCCGgCTTTaTCGATGgcCGTCTTGACGTCGpNSB651pNSB654M3–2CTTTGTCGATcgcCGTCTTGACGTCGpNSB687pNSB683M3–3GAGTATCTCCGgCTTTaTCGATGTACGpNSB688pNSB684M3–4TACAAGAGTAgCTCCGgCTTTcgCGATGTACGTCTTGACpNSB741pNSB747M3–5CTCCGTCTTTaTCGATGaACGTCTTGACpNSB781pNSB779M3–6GAGTATCTCCGTCTgcaTCGATGTACGpNSB782pNSB780 Open table in a new tab Wild type and mutant GST-AL1 fusion proteins were expressed in Spodoptera frugiperda (Sf9) cells using a Tn7-based baculovirus expression system (40Luckow V.A. Lee S.C. Barry G.F. Olins P.O. J. Virol. 1993; 67: 4566-4579Crossref PubMed Google Scholar) and purified from Sf9 cell cultures by glutathione affinity chromatography according to published protocols (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). Purified proteins were visualized by electrophoresis in 16% polyacrylamide-SDS gels and staining with Coomassie Brilliant Blue dye. Interactions between authentic AL1 and mutant GST-AL1 proteins were assayed by copurification on glutathione-Sepharose, followed by immunoblot analysis using AL1 polyclonal antisera (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar). DNA gel shift assays were performed as described previously (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). An 83-base pair EcoRI fragment containing the AL1 DNA binding motif (TGMV A positions 28–84) was isolated from pNSB378 and 3′ end-labeled using Klenow and [α-32P]dATP. The radiolabeled DNA was incubated with purified GST-AL1 fusion proteins for 1 h at room temperature at the concentrations indicated in the figure legends. The bound and free probe were resolved on 1% agarose gels, dried onto DE-81 paper, and analyzed by autoradiography. For DNA cleavage and ligation, oligonucleotides corresponding to sequences in the hairpin of the TGMV (+)-strand origin were 5′ end-labeled using polynucleotide kinase and [γ-32P]ATP. The oligonucleotides CR13 (5′-GTTTAATATTACCGGATGGCCGC) and CR33 (5′-GCGGCCATCCGTTTAATATT) were used for assays with full-length mutant and wild type AL1 proteins. For assays with truncated AL1 proteins, only one oligonucleotide, CR13 or CR34 (5′-GCGGCCATCCGTTTAATATTACCGGATGG) was radiolabeled and the cold oligonucleotide was titrated into the reactions as described in the figure legend. Approximately 5000 cpm of each labeled DNA was incubated with ∼100 ng of purified GST-AL1 fusion protein in 10 μl of cleavage buffer (25 mm Tris-HCl, pH 7.5, 75 mmNaCl, 5 mm MgCl2, 2.5 mm EDTA, 2.5 mm dithiothreitol, and 250 ng of poly(dI-dC)) at 37 °C for 30 min. The reactions were terminated by adding 6 μl of gel loading buffer (95% formamide, 20 mm EDTA, 0.05% bromphenol blue) and heating to 90 °C for 2 min. The reaction products were resolved on 15% polyacrylamide denaturing gels and analyzed by autoradiography. Protoplasts were isolated fromNicotiana tabacum NT-1 suspension cells, electroporated, and cultured according to published methods (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). The transfections contained 15 μg each of replicon DNA containing a partial tandem copy of TGMV B (pTG1.4B described in Ref. 14Fontes E.P.B. Gladfelter H.J. Schaffer R.L. Petty I.T.D. Hanley-Bowdoin L. Plant Cell. 1994; 6: 405-416Crossref PubMed Scopus (171) Google Scholar), wild type or mutant AL1 expression cassette, and an AL3 plant expression cassette (pNSB46 described in Ref. 11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). Total DNA was extracted 3 days after transfection and analyzed for double-stranded viral DNA accumulation by DNA gel blot hybridization (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). Two key steps in initiation of RCR are origin recognition and generation of a free 3′-OH for priming plus-strand DNA synthesis. TGMV AL1 mediates both of these processes by binding double-stranded DNA in a sequence-specific manner and by cleaving at a unique site in the origin. Earlier studies established that the AL1 N terminus is necessary for both activities (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This region of AL1 contains several conserved sequence and structural motifs (Fig. 1), including a highly predicted pair of α-helices between amino acids 25 and 52 (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The sequences of both helices are strongly conserved among dicot-infecting geminiviruses, and the second helix is amphipathic in character. This conservation suggested that the predicted α-helices may be necessary for AL1 function. To test this hypothesis, we generated four site-directed mutants of TGMV AL1 that are modified in either helix 1 or helix 2 (Fig. 1 B) and compared their activities to the wild type protein in vivo and in vitro (Fig. 1 B). The helix mutants were first tested for the ability to direct replication of TGMV B DNA in tobacco protoplasts with wild type TGMV AL3 also supplied in trans. Helix 1-mutant 2 (H1–2), which contains a S25V change and resembles the AL1 protein of the closely related geminivirus BGMV, supported wild type levels of TGMV B replication (Fig. 2 A, cf. lanes 1 and 2). In contrast, H1–1, which has alanine substitutions at three conserved charged residues in helix 1, failed to support viral DNA replication (Fig. 2 A, lane 3). The mutations in helix 2 either converted three conserved charged residues to alanine (H2–1), included a I45G change (H2–2), or altered the helix to resemble BGMV AL1 (H2–3). All helix 2 mutations abolished transient replication (Fig. 2 A, lanes 3–5). The mutant phenotypes established that, in general, the amino acid sequences of the predicted helices are essential for AL1 activity in vivo. The negative effect of the I45G replacement in H2–2, which should disrupt helix 2, suggested that the structure is also required for AL1 function. The AL1 helix mutants defective in replication assays were expressed as GST-AL1 fusion proteins in insect cells, purified by glutathione affinity chromatography, and examined for various AL1 activitiesin vitro. H1–2 was not included in these experiments because of its wild type replication phenotype. The mutant proteins were tested for DNA binding activity in gel shift assays using a radiolabeled double-stranded DNA probe that includes the TGMV AL1 DNA binding site. None of the helix mutants bound DNA (Fig. 2 B, lanes 3–6), even though wild type AL1 efficiently shifted the probe in a parallel reaction (lane 2). The mutant AL1 proteins were also tested for DNA cleavage activity using a radiolabeled, single-stranded oligonucleotide corresponding to the loop and right side of the hairpin in the plus-strand origin. Wild type GST-AL1 cleaved the 23-nt substrate to give a 10-nt radiolabeled product (Fig. 2 C, lane 2). Mutants H1–1 (Fig. 2 C, lane 3) and H2–3 (lane 6) displayed severely attenuated DNA cleavage activity, whereas H2–1 (lane 4) and H2–2 (lane 5) had no detectable activity. These results demonstrated that the predicted helices 1 and 2 are required both for DNA binding and cleavage by TGMV AL1. Helices 1 and 2 are outside of the AL1 interaction domain, and their mutation should have no effect on oligomerization if the proteins are properly expressed and folded. To verify that the failure of the AL1 helix mutants to bind and cleave DNA was not due to global misfolding, the proteins were assayed for their abilities to form oligomers with authentic AL1. Wild type GST-AL1 (Fig. 3 A, lane 1) and GST fusions of H1–1 (lane 2), H2–1 (lane 3), H2–2 (lane 4), and H2–3 (lane 5) were coexpressed with authentic AL1 in insect cells, as determined by immunoblotting of total protein extracts. Like wild type GST-AL1 (Fig. 3 A, lane 6), all of the mutant proteins copurified with authentic AL1 on glutathione-Sepharose (lanes 7–10), indicating that the helix mutations did not impair AL1/AL1 interactions and, instead, specifically affected the DNA binding and cleavage activities of AL1. The N terminus of AL1 also includes three conserved sequence motifs that are found in many RCR initiator proteins (Fig. 1; Refs. 35Koonin E.V. Ilyina T.V. J. Gen. Virol. 1992; 73: 2763-2766Crossref PubMed Scopus (132) Google Scholar and 36Ilyina T.V. Koonin E.V. Nucleic Acids Res. 1992; 20: 3279-3285Crossref PubMed Scopus (508) Google Scholar). Laufset al. (27Laufs J. Schumacher S. Geisler N. Jupin I. Gronenborn B. FEBS Lett. 1995; 377: 258-262Crossref PubMed Scopus (67) Google Scholar) showed that motif 3 corresponds to the endonucleolytic active site, but the roles of motifs 1 and 2 in RCR have not been investigated. We specifically modified these motifs in TGMV AL1 and analyzed the impact of the mutations on protein function (Fig. 1 B). In M1–1, all four motif I residues (F16LTY19) were replaced with alanine. In M1–2 and M1–3, individual aromatic residues were altered to give F16A and Y19A, respectively. The motif 2 mutant contained alanine substitutions for the core HLH sequence. Transient replication assays revealed that none of the motif 1 or 2 mutants (Fig. 4 A, lanes 2–5) supported TGMV B amplification in tobacco protoplasts, establishing that both motifs are essential for AL1 activity in vivo. To gain insight into the biochemical basis of the mutant replication phenotypes, we purified GST-AL1 fusion proteins corresponding to the motif 1 and 2 mutants from insect cells and tested them for DNA binding and cleavage in vitro. All of the mutant proteins failed to bind double-stranded DNA (Fig. 4 B, lanes 2–5). Similarly, none of the mutants had detectable single-stranded DNA cleavage activity (Fig. 4 C, lanes 2–5). In parallel assays, wild type GST-AL1 (lane 1) efficiently bound (Fig. 4 B) and cleaved (Fig. 4 C) the respective DNA probes. Protein interaction experiments (Fig. 3 B) established that M1–1 (lanes 3 and 8) and M2–1 (lanes 4 and 9) can form oligomers with wild type AL1 and, thus, are not globally misfolded. Together, these results showed that motifs 1 and 2 of TGMV AL1 are required for both DNA binding and cleavage during initiation of RCR. Motif 3 includes several conserved amino acids that may contribute to AL1 function. We generated six TGMV AL1 mutants that modified one or more residues in motif 3 and analyzed their activities in vivo and in vitro. In M3–1, alanines were substituted for the catalytic Tyr-103 and conserved Asp-107 residues. As expected, M3–1 did not support TGMV B replication in tobacco protoplasts (Fig. 5 A, lane 2) and was defective for DNA cleavage (Fig. 5 C, lane 2). Gel shift assays with M3–1 revealed that double-stranded DNA binding was also attenuated over a range of protein concentrations (Fig. 5 B, lanes 5–7), even though wild type GST-AL1 readily formed protein/DNA complexes at the same concentrations (lanes 2–4). In some experiments, low levels of DNA binding were observed with the M3–1 protein (data not shown). Protein interaction experiments showing that M3–1 can oligomerize with wild type AL1 (Fig. 3 B, lanes 5 and 10) indicated that the motif 3 mutant is not globally misfolded. The DNA binding defect of M3–1 was unexpected because of the direct involvement of motif 3 in catalysis of DNA cleavage. To better characterize the role of motif 3 and specifically Tyr-103 in DNA binding, we generated two different mutations at this position. In M3–2, Tyr-103 was changed to alanine, while M3–5 contained a phenylalanine substitution. Like M3–1, neither M3–2 or M3–5 supported viral DNA replication (Fig. 5 A, lanes 3 and 6) or cleaved DNA (Fig. 5 C, lanes 3 and 6), confirming that Tyr-103 is necessary for these processes. However, only M3–2 was impaired for DNA binding over a range of protein concentrations (Fig. 5 B, lanes 5–7), whereas M3–5 displayed wild type DNA binding properties (lanes 17–19). These results demonstrated that Tyr-103 contributes to both the DNA bind
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