@article{orozco_kong_batts_elledge_hanley-bowdoin_2000, title={The multifunctional character of a geminivirus replication protein is reflected by its complex oligomerization properties}, volume={275}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.275.9.6114}, abstractNote={Tomato golden mosaic virus (TGMV), a member of the geminivirus family, encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its plant host. TGMV AL1 is an oligomeric protein that binds double-stranded DNA and catalyzes cleavage and ligation of single-stranded DNA. The oligomerization domain, which is required for DNA binding, maps to a region that displays strong sequence and structural homology to other geminivirus Rep proteins. To assess the importance of conserved residues, we generated a series of site-directed mutations and analyzed their impact on AL1 function in vitro and in vivo. Two-hybrid experiments revealed that mutation of amino acids 157–159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability. Changes at positions 157–159 also disrupted interaction between the full-length mutant protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells. The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL1 complexes can be stabilized by amino acids outside of the oligomerization domain. Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA replication. In contrast, the same mutants were enhanced for AL1-mediated transcriptional repression. The replication-defective AL1 mutants also interfered with replication of a TGMV A DNA encoding wild type AL1. Full-length mutant AL1 was more effective in the interference assays than truncated proteins containing the oligomerization domain. Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein. Tomato golden mosaic virus (TGMV), a member of the geminivirus family, encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its plant host. TGMV AL1 is an oligomeric protein that binds double-stranded DNA and catalyzes cleavage and ligation of single-stranded DNA. The oligomerization domain, which is required for DNA binding, maps to a region that displays strong sequence and structural homology to other geminivirus Rep proteins. To assess the importance of conserved residues, we generated a series of site-directed mutations and analyzed their impact on AL1 function in vitro and in vivo. Two-hybrid experiments revealed that mutation of amino acids 157–159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability. Changes at positions 157–159 also disrupted interaction between the full-length mutant protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells. The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL1 complexes can be stabilized by amino acids outside of the oligomerization domain. Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA replication. In contrast, the same mutants were enhanced for AL1-mediated transcriptional repression. The replication-defective AL1 mutants also interfered with replication of a TGMV A DNA encoding wild type AL1. Full-length mutant AL1 was more effective in the interference assays than truncated proteins containing the oligomerization domain. Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein. tomato golden mosaic virus activation domain glutathioneS-transferase maize streak virus Geminiviruses are a large family of plant viruses with circular, single-stranded DNA genomes that replicate in the nuclei of infected cells (reviewed in Ref. 1.Hanley-Bowdoin L. Settlage S.B. Orozco B.M. Nagar S. Robertson D. CRC Crit. Rev. Plant Sci. 1999; 18: 71-106Crossref Google Scholar). The single-stranded genome is converted to a double-stranded DNA that serves as the template for rolling circle replication (2.Saunders K. Lucy A. Stanley J. Nucleic Acids Res. 1991; 19: 2325-2330Crossref PubMed Scopus (167) Google Scholar, 3.Stenger D.C. Revington G.N. Stevenson M.C. Bisaro D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8029-8033Crossref PubMed Scopus (288) Google Scholar, 4.Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (88) Google Scholar) and transcription (5.Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar, 6.Sunter G. Bisaro D.M. Virology. 1989; 173: 647-655Crossref PubMed Scopus (47) Google Scholar). Geminiviruses do not encode their own polymerases and, instead, rely on host enzymes for viral DNA and RNA synthesis. These characteristics make geminiviruses excellent model systems for studying plant DNA replication and transcription mechanisms. The geminivirus, tomato golden mosaic virus (TGMV),1 has a bipartite genome that encodes seven open reading frames that are divergently transcribed. The 5′-intergenic region separating the transcription units is nearly identical between the two DNA components and includes the plus strand origin of replication (7.Lazarowitz S.G. Wu L.C. Rogers S.G. Elmer J.S. Plant Cell. 1992; 4: 799-809Crossref PubMed Scopus (116) Google Scholar, 8.Orozco 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). The promoter for complementary sense transcription overlaps the replication origin (5.Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar,9.Hanley-Bowdoin L. Elmer J.S. Rogers S.G. Plant Cell. 1989; 1: 1057-1067PubMed Google Scholar) and shares some of the cis-elements involved in origin function (10.Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar). A directly repeated sequence, GGTAG, is required for origin recognition (11.Fontes 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) and transcriptional repression of the complementary sense (AL1) promoter (10.Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar). Similarly, the TATA-box and G-box transcription factor binding sites in the AL1 promoter act as replication enhancer elements (12.Eagle P.A. Hanley-Bowdoin L. J. Virol. 1997; 71: 6947-6955Crossref PubMed Google Scholar). In contrast, three elements in the TGMV intergenic region are necessary for origin function but have little or no effect on AL1 promoter activity. A hairpin structure with a 9-base pair loop sequence that is conserved among all geminiviruses is essential for replication and contains the cleavage site for initiating plus strand DNA synthesis (4.Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (88) Google Scholar, 13.Laufs 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 (259) Google Scholar, 14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). A conserved sequence between the AL1 binding site and the hairpin, the AG-motif, is also required for replication (8.Orozco 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). The third element, the CA motif, is located outside of the minimal origin but its deletion reduced replication 20-fold (8.Orozco 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). The role of the AG- and CA-motifs in TGMV origins is not known, but one possibility is that they bind host factors that facilitate initiation of plus strand DNA replication. 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 (15.Elmer 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, 16.Sunter G. Hartitz M.D. Hormuzdi S.G. Brough C.L. Bisaro D.M. Virology. 1990; 179: 69-77Crossref PubMed Scopus (167) Google Scholar) (AL1 homologues are also designated C1 or Rep.) AL1 is a multifunctional protein that mediates both virus-specific recognition of its cognate origin (17.Fontes 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) and transcriptional repression by binding to the directly repeated sequence in the intergenic region (10.Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar, 12.Eagle P.A. Hanley-Bowdoin L. J. Virol. 1997; 71: 6947-6955Crossref PubMed Google Scholar). AL1 initiates and terminates plus strand replication (13.Laufs 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 (259) Google Scholar, 14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar, 18.Heyraud-Nitschke F. Schumacher S. Laufs J. Schaefer S. Schell J. Gronenborn B. Nucleic Acids Res. 1995; 23: 910-916Crossref PubMed Scopus (142) Google Scholar) and induces the accumulation of a host replication factor, proliferating cell nuclear antigen, in infected cells (19.Nagar S. Pedersen T.J. Carrick K. Hanley-Bowdoin L. Robertson D. Plant Cell. 1995; 7: 705-719Crossref PubMed Scopus (156) Google Scholar). Recombinant AL1 specifically binds double-stranded DNA (11.Fontes 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, 20.Fontes E.P.B. Luckow V.A. Hanley-Bowdoin L. Plant Cell. 1992; 4: 597-608Crossref PubMed Scopus (136) Google Scholar), cleaves and ligates single-stranded DNA in the invariant sequence of the hairpin loop (14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar, 21.Laufs J. Schumacher S. Geisler N. Jupin I. Gronenborn B. FEBS Lett. 1995; 377: 258-262Crossref PubMed Scopus (67) Google Scholar), and hydrolyzes ATP (22.Desbiez 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, 23.Orozco 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 also interacts with itself (23.Orozco 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 viral replication enhancer protein AL3 (24.Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar), and a maize homologue of the cell cycle regulatory protein, retinoblastoma (25.Ach 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). We previously mapped the TGMV AL1 domains for double-stranded DNA binding, single-stranded DNA cleavage and ligation, and AL1 oligomerization (23.Orozco 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, 26.Orozco B.M. Hanley-Bowdoin L. J. Biol. Chem. 1998; 273: 24448-24456Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The DNA cleavage/ligation domain was located to the first 120 amino acids, and the oligomerization domain was mapped between amino acids 120 and 181. DNA binding activity required amino acids 1–130 for protein-DNA contacts and the AL1 oligomerization domain. Because of its importance in AL1 function, we have further characterized the sequences required for oligomerization in vitro and assessed the impact of site-directed mutations in the domain in vivo. Constructs are listed in Table I. The plasmid pNSB148, which contains the AL1 coding sequence in a pUC118-background, was used as the template for site-directed mutagenesis (27.Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 482-492Google Scholar). The oligonucleotide primers and resulting clones are also listed in TableI. DNA fragments containing the mutations were verified by DNA sequence analysis. Plant expression cassettes for mutant AL1 proteins were generated by subcloningSalI/NcoI fragments (TGMV A position 2442 and 2059) from the mutant clones into the same sites in the wild type AL1 plant expression cassette pMON1549 (11.Fontes 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 35S promoter with a duplicated enhancer region and the pea E9 rbcS 3′-end.Table IAL1 mutationsMutationOligonucleotideBaculovirus vectorYeast GAL4-ADPlant expressionWild typeN/ApMON1680pNSB809pMON1549FQ118CACTTCGACCGTCGACCGCGGCTTCTCCCCAN/ApNSB872pNSB866D120CACTTCGGCCGGCGACCGCGGCTTCTCCCCAN/ApNSB871pNSB865RS-R125GCAACCTCCTgcAGCggccgcACCGTCGACCTGGAN/ApNSB786pNSB695QT130CAGCGTCGTTgctaGcTgcGCAACCTCCTCTAGCAN/ApNSB788pNSB696ND133CTGCTGCAGCGgCcgcAGATGTTTGGCAApNSB603PNSB790pNSB670E–N140GGAAGAAGCAgcTAACGCggCcGCTGCAGCGTCGTpNSB604PNSB893pNSB640KEE146TCTGCAGGGCTgCggCcgcGGAAGAAGCATTTAApNSB605PNSB894pNSB641REK154TTCTGGGATTgcggCcgcAATTATCTGCAGGGpNSB606pNSB759pNSB671EKY159GAACTGAAATAAAgcggccgCTGGGATTTTCTCTCpNSB607pNSB760pNSB672Q-HN165GCTATTTAGAgcGgcGAACgcAAATAAATATTTTTCTGGGATpNSB608pNSB761pNSB698N-DR172ATCAAATATCgcAgCTAgcgcGCTATTTAGATTGTGpNSB609pNSB762pNSB707K–E179GAAGCCATGGcgCcGGAGTCgcATCAAATATCCpNSB610pNSB763pNSB697 Open table in a new tab Baculovirus vectors were generated for expression of mutant and truncated AL1 proteins in insect cells. Expression vectors coding for mutant AL1 proteins were made by subcloningBglII/BamHI inserts from the mutant plant expression cassettes into the BamHI site of pMON27025 (28.Luckow V.A. Lee S.C. Barry G.F. Olins P.O. J. Virol. 1993; 67: 4566-4579Crossref PubMed Google Scholar). Expression vectors for the truncated proteins, AL1119–352(pNSB516), AL11–120 (pNSB388), and AL11–180(pNSB517), have been described previously (23.Orozco 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 N-terminal truncations, AL1134–352 and AL1147–352, were generated by inserting a double-stranded oligonucleotide containing anSphI site into the NotI site of pNSB593 and pNSB595 to create in-frame start codons. AL1160–352 was created by inserting an SphI linker with a start codon (New England Biolabs, Beverly, MA) into the SspI site of pMON1539 (29.Hanley-Bowdoin L. Elmer J.S. Rogers S.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1446-1450Crossref PubMed Scopus (84) Google Scholar). SphI/BamHI fragments from the resulting clones were inserted into the same sites of the baculovirus vector, pNSB448 (23.Orozco 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), to give pNSB803 (AL1134–352), pNSB876 (AL1147–352), and pNSB633 (AL1160–352). The C-terminal truncation, AL11–158 (pNSB646), was generated by digesting pMON1539 with NdeI and SspI, repairing with Klenow, and subcloning into the filled BamHI site of pMON27025 to create an in-frame stop codon. The AL11–168 truncation, pNSB708, was created by inserting anXbaI linker into the repaired BssHII of pNSB609, generating an in-frame stop codon. Yeast expression cassettes were generated using the pAS1–1 and pACT2 vectors from CLONTECH (Palo Alto, CA). TheBamHI/NdeI fragment of pMON1539 was cloned into the same sites of pAS2–1 to give pNSB736, which contained the GAL4 DNA binding domain fused to wild type AL1 sequences. The ends of the sameBamHI/NdeI fragment were repaired with Klenow and cloned into the SmaI site of pACT2 to give pNSB735. TheAatII/BamHI fragment of pNSB735 was then replaced with the AatII/BamHI fragment from pMON1549. The resulting clone, pNSB809, contained the GAL4 activation domain (AD) fused to wild type AL1 sequences. Mutant AL1 yeast expression cassettes were created by replacing the wild typeAatII/BamHI fragment of pNSB735 with mutantAatII/BamHI fragments from the corresponding plant expression cassettes. Protoplasts were isolated from Nicotiana tabacum (BY-2) orNicoxiana benthamiana suspension cells, electroporated, and cultured according to published methods (11.Fontes 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). For replication assays, N. tabacum transfections included 15 μg each of replicon DNA containing a partial tandem copy of TGMV B (pTG1.4B, Ref. 17.Fontes 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 plant expression cassette, and an AL3 plant expression cassette (pNSB46, Ref. 11.Fontes 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). For the interference assays, 2 μg of replicon DNA containing a partial tandem copy of TGMV A (pMON1565, Ref. 14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar) was cotransfected with 40 μg of mutant AL1 expression cassette or the empty expression vector (pMON921, Ref. 11.Fontes 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 post-transfection and analyzed for double- and single-stranded viral DNA accumulation by DNA gel blot hybridization (11.Fontes 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 viral DNA was quantified by phosphorimager analysis in a minimum of three independent experiments. For transcriptional repression assays, N. benthamianaprotoplasts were transfected with 15 μg of luc reporter construct (pNSB114), 15 μg of AL1 expression cassette, and 36 μg of sheared salmon sperm DNA (10.Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar). Luciferase activity in total soluble protein extracts was measured 36 h post-transfection and standardized for protein concentration. Repression was determined as the ratio of Luc activity in the absence versus the presence of AL1. Each expression cassette was assayed in triplicate in at least three independent experiments. Recombinant proteins were produced inSpodoptera frugiperda Sf9 cells using a baculovirus expression system according to published protocols (14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar, 24.Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar). Protein extracts from cells co-expressing authentic and GST-AL1 fusion proteins were assayed for AL1 oligomerization by co-purification on glutathione-Sepharose (24.Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar). Co-purification was monitored by SDS-polyacrylamide electrophoresis followed by transfer to nitrocellulose membrane (Schleicher and Schuell) and immunoblotting using the ECL detection system (Amersham Pharmacia Biotech). Primary antibodies were rabbit polyclonal anti-GST (Upstate Biotechnology Inc.) and anti-AL1 antisera (24.Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar). The Saccharomyces cerevisiae strain Y187 (MATα,ura3–52, his3–200, ade 2–101,trp 1–901, leu 2–3, 112,gal4Δ, met −, gal80Δ,URA3::GAL1 UAS-GAL1 TATA-lacZ) was transformed using lithium acetate/polyethylene glycol (30.Geitz D. St. Jean A. Woods R.A. Schiesti R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2895) Google Scholar). The DNAs were pNSB736, which expresses the GAL4 binding domain-wild type AL1 fusion, and either pNSB809, which produces the GAL4 AD-wild type AL1 protein, or the equivalent cassettes corresponding to the GAL4 AD-AL1 mutants. For β-galactosidase assays, yeast transformants were grown to an A 600 of 0.5 in 3 ml of synthetic dropout medium lacking tryptophan and leucine (31.Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar). Yeast were pelleted at 1000 × g for 5 min, rinsed with Z buffer (0.1m NaPO4, pH 7, 10 mm KCl, 1 mm MgSO4, 40 mmβ-mercaptoethanol) (31.Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar), and resuspended in 300 μl of Z buffer. The cells were subjected to three freeze/thaw cycles in liquid nitrogen and centrifuged at 5000 × g for 2 min. The supernatant (150 μl) was assayed for β-galactosidase activity in a total reaction volume of 250 μl using the substrateo-nitrophenyl β-d-galactopyranoside, as described by CLONTECH. Accumulation of theo-nitrophenol product was measured atA 420 using a BioKinetics microplate reader (Bio-Tek Instrument Inc., Winooski, VT). Protein concentrations were measured by Bradford assays (Bio-Rad). The enzyme-specific activity (1 unit = 1.0 μm product/min at pH7.3 at 37 °C) resulting from interaction between two-hybrid cassettes carrying wild type AL1 sequences was determined using purified β-galactosidase (Sigma) as the standard. The relative activities of the mutants were normalized against the wild type AL1 interaction level, which was set to 100%. The β-galactosidase specific activity for wild type and mutant AL1 proteins was adjusted for background from pNSB736 alone. The different constructs were tested in a minimum of two experiments, each of which assayed four independent transformants for each construct. For immunoblot analysis, individual yeast transformants were grown in 5 ml of medium containing 1% yeast extract, 2% bacto-peptone, 2% glucose, pH 5.8 (31.Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar) to an A 600 of 1. An equal volume of crushed ice was added and the culture was centrifuged at 1000 × g for 5 min. The resulting pellet was washed once with ice cold water and resuspended in 80 μl of modified radioimmune precipitation buffer (150 mm NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 50 mmTris-HCl, pH 7.5 (32.Harlow E. Lane D. Antibodies, A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1988Google Scholar), containing 1% (w/v) SDS) and protease inhibitors (6 μg/ml pepstatin A, 10 μg/ml leupeptin, 20 μg/ml aprotinin, 8 mm benzamidine, 1 mmphenylmethylsulfonyl fluoride). Glass beads (40 μl, 425–600 μm, Sigma) were added and the sample was vortexed at maximum speed for four 30-s intervals separated by 2-min intervals on ice. The sample was then centrifuged at 5000 × g for 2 min at 4 °C. The supernatant was recovered, and the protein concentration was determined using Bradford assays. Total protein (100 μg) was resolved on 12% polyacrylamide/SDS gels and analyzed by immunoblotting using a GAL4 AD monoclonal antibody at 0.4 μg/ml (CLONTECH). The domains for TGMV AL1 DNA binding and DNA cleavage/ligation activities are well defined, and key structural and sequence motifs have been identified for these functions (23.Orozco 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, 26.Orozco B.M. Hanley-Bowdoin L. J. Biol. Chem. 1998; 273: 24448-24456Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), whereas the AL1 oligomerization domain has only been broadly located to the center of the protein (23.Orozco 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). In this paper, closely spaced N- and C-terminal truncations were generated to define the limits of the AL1 oligomerization domain (Fig.1 A). A GST fusion corresponding to full-length AL1 (GST-AL1) was co-expressed with the truncated AL1 proteins in baculovirus-infected insect cells, and protein complexes were purified on glutathione-Sepharose resin. Total extracts and purified proteins were resolved by SDS-polyacrylamide gel electrophoresis, and proteins were visualized by immunoblotting with AL1 and GST polyclonal antisera. As reported previously (23.Orozco 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 C-terminal truncation AL11–180 (Fig. 1 B,lanes 3 and 6) copurified with full-length GST-AL1. Further deletion of the C terminus to amino acid 168 (lanes 2 and 5) or 158 (lanes 1 and4) abolished interactions with GST-AL1, demonstrating that the C-terminal limit of the oligomerization domain is between position 168 and 180. The N-terminal truncations to amino acids 134 (Fig.1 C, lanes 3 and 6), 147 (lanes 2 and 5), and 160 (lanes 1 and 4) showed a gradual disappearance of interaction with GST-AL1. AL1134–352 consistently displayed interactions with GST-AL1, whereas AL1147–352 and AL1160–352interactions varied between weak to background levels, making it more difficult to define the N-terminal limit. Contacts outside the oligomerization domain may contribute to complex stability or multimerization and account for the low level interactions observed with AL1147–352 and AL1160–352. To address this possibility, we asked whether full-length AL1 copurified when only the oligomerization domain was fused to GST (GST-AL1119–180). Full-length AL1 interacted with GST-AL1119–180 but not with GST alone (Fig.2 B, lanes 1 and2), demonstrating that amino acids 119–180 are sufficient for oligomerization. We then examined the abilities of the N-terminal AL1 truncations to bind GST-AL1119–180. In this assay, deletion to positions 119 (Fig. 1 D, lanes 1 and5) and 134 (lanes 2 and 6) did not affect oligomerization, whereas further deletion to positions 147 (lanes 3 and 7) and 160 (lanes 4 and8) abolished interactions with GST-AL1119–180. Together, these results showed that AL1 amino acids 134–180 contain the oligomerization domain and that sequences outside the domain contribute stabilizing contacts. Alanine substitutions were generated in conserved charged or hydrophobic residues within the oligomerization domain to identify key amino acids that contribute to AL1 interactions (Fig.3, the mutations are designated by the corresponding wild type sequence and the position of the last amino acid that was altered; dashes indicate amino acids that were not changed). Alanine was selected because it is structurally neutral and should not interfere with normal protein folding. The mutations are within a region that includes a pair of predicted α-helices (26.Orozco B.M. Hanley-Bowdoin L. J. Biol. Chem. 1998; 273: 24448-24456Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and downstream sequences required for oligomerization. All of the mutant AL1 proteins co-purified with GST-AL1 on glutathione resin when co-expressed in insect cells (Fig. 2 A), showing that they formed stable complexes with the wild type protein. Similar results were observed when both the test and GST fusion proteins carried the mutations (data not shown). In contrast, when interactions between the mutant AL1 proteins and GST-AL1119–180 were examined mutant EKY159 (Fig. 2 B, lane 6) was defective for AL1 interactions. Thus, sequences outside of the oligomerization domain in the full-length AL1 masked the effect of the EKY159 mutation in Fig.2 A, which is consistent with our previous conclusion that sequences outside the oligomerization domain stabilize AL1 interactions. The oligomerization mutant, EKY159, was assayed with truncated GST-AL1 proteins to locate the stabilizing region. As shown above, EKY159 bound full-length GST-AL1 (Fig. 2 C,lanes 1 and 5) but not GST-AL1119–180 (lanes 4 and 8). EKY159 also bound an N-terminal truncation, GST-AL1119–352(lanes 3 and 7), whereas no interaction was detected with a C-terminal truncation, GST-AL11–180(lanes 2 and 6), demonstrating that the AL1 C terminus contributes stabilizing contacts. The quantitative impact of each mutation on AL1 oligomerization was measured using a yeast two-hybrid system. Expression cassettes for wild type or mutant AL1 fused to the GAL4 AD were cotransformed into yeast with a cassette for wild type AL1 fused to the GAL4 DNA binding domain. Activation of a GAL4-responsive promoter driving a lacZreporter was assayed by measuring β-galactosidase activity in total soluble protein extracts. In extracts from yeast cotransfected with AD and binding domain cassettes carrying wild type AL1 sequences, 142 mu/mg total protei}, number={9}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Orozco, BM and Kong, LJ and Batts, LA and Elledge, S and Hanley-Bowdoin, L}, year={2000}, month={Mar}, pages={6114–6122} }
 @article{gladfelter_eagle_fontes_batts_hanley-bowdoin_1997, title={Two domains of the AL1 protein mediate geminivirus origin recognition}, volume={239}, ISSN={["0042-6822"]}, DOI={10.1006/viro.1997.8869}, abstractNote={The geminiviruses tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) have bipartite genomes. Their A and B DNA components contain cis -acting sequences that function as origins of replication, while their A components encode the trans -acting replication proteins—AL1 and AL3. Earlier experiments demonstrated that virus-specific interactions between the cis - and trans -acting functions are required for TGMV and BGMV replication and that the AL1 proteins of the two viruses specifically bind their respective origins. In the current study, characterization of AL1 and AL3 proteins produced from plant expression cassettes in transient replication assays revealed that interaction between AL1 and the origin is responsible for virus-specific replication. The AL3 protein does not contribute to specificity but can be preferred by its cognate AL1 protein when replication is impaired. Analysis of chimeric proteins showed that two regions of AL1 act as specificity determinants during replication. The first domain is located between amino acids 1 and 116 and recognizes the AL1 origin binding site. The second region, which is between amino acids 121 and 209, is not dependent on the known AL1 DNA binding site. Analysis of wild type and chimeric proteins in transient transcription assays showed that AL1 also represses its own promoter in a virus-specific manner. Transcriptional specificity is conferred primarily by AL1 amino acids 1–93 with amino acids 121–209 making a smaller contribution. Together, these results demonstrated that the virus-specific interactions of AL1 during replication and transcription are complex, involving at least two discreet domains of the protein.}, number={1}, journal={VIROLOGY}, author={Gladfelter, HJ and Eagle, PA and Fontes, EPB and Batts, L and Hanley-Bowdoin, L}, year={1997}, month={Dec}, pages={186–197} }