Conserved Sequence and Structural Motifs Contribute to the DNA Binding and Cleavage Activities of a Geminivirus Replication Protein

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 (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. Hanley-Bowdoin L. Plant Cell. 1994; 6: PubMed Scopus Google Scholar, D.C. Virology. PubMed Scopus Google Scholar, H.J. Eagle P.A. E.P.B. Hanley-Bowdoin L. Virology. PubMed Scopus (47) Google Scholar). The IR also a with a pair sequence conserved among geminiviruses that is and of F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (90) Google Scholar, J. F. Matzeit V. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, B.M. Hanley-Bowdoin L. J. Google Scholar). experiments established that the is essential for TGMV replication B.M. Hanley-Bowdoin L. J. Google Scholar). TGMV encodes two AL1 and that are required for viral AL1 is necessary for viral DNA by mechanism L. G. Gardiner W.E. Bisaro D.M. Nucleic Acids Res. PubMed Scopus Google Scholar, G. Bisaro D.M. Virology. PubMed Scopus (167) Google Scholar). AL1 is a multifunctional protein that virus-specific recognition to its plus-strand origin D.C. Virology. PubMed Scopus Google Scholar, H.J. Eagle P.A. E.P.B. 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Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar, E.P.B. Luckow V.A. Hanley-Bowdoin L. Plant Cell. PubMed Scopus Google Scholar) and single-stranded DNA in the sequence of the B.M. Hanley-Bowdoin L. J. Google Scholar). of the protein of tomato yellow leaf curl virus a TGMV AL1 that to the 5′ of the DNA by a and catalyzes ligation of the cleavage J. S. Gronenborn B. PubMed Scopus Google Scholar). has also been demonstrated for the proteins from and TGMV A. J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). TGMV AL1 is in protein interactions. forms B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), binds S.B. B. Hanley-Bowdoin L. J. PubMed Google Scholar), and with a of the protein, Hanley-Bowdoin L. Cell. Biol. PubMed Scopus Google Scholar). The protein from virus also with proteins from and EMBO J. PubMed Scopus Google Scholar, S. M. Virology. PubMed Scopus Google Scholar, EMBO J. PubMed Scopus Google Scholar). In study, we used truncated proteins in a to the of TGMV AL1 that are for DNA DNA binding and oligomerization B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These experiments showed that the DNA cleavage domain is located in the amino acids of AL1 the oligomerization domain maps to the protein between amino acids and DNA binding a region that overlaps the DNA cleavage and oligomerization domains. on these we that amino acids between and 181 are necessary for binding that AL1 is required for DNA In this study, we 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 on amino acid are conserved among geminivirus proteins and initiator proteins from other systems J. PubMed Scopus Google Scholar, Nucleic Acids Res. PubMed Scopus Google Scholar), and on a predicted in the N of proteins of dicot-infecting geminiviruses B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We also used a heterologous protein domain to the of oligomerization for binding and DNA The the AL1 sequence in a was used as the template for site-directed Proc. Natl. Acad. Sci. U. S. A. Scholar). The and are in The of the mutations and used for were by DNA sequence Plant with the mutant AL1 were generated by amino acids 1–120) from the mutant the in a AL1 plant (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 AL1 is the of the mosaic virus promoter with a R. A. M. J. PubMed Scopus Google Scholar) and the from the gene G. R. EMBO J. PubMed Scopus Google Scholar). for AL1 proteins fused to a glutathione S-transferase were generated by the mutant plant with and and the with DNA The AL1 were the of The the by a a cleavage and a for proteins B.M. Hanley-Bowdoin L. J. Google Scholar). The mutant are in in a and mutant proteins were in cells a V.A. J. 1993; PubMed Google Scholar) and from by glutathione to B.M. Hanley-Bowdoin L. J. Google Scholar). proteins were by in and with between AL1 and mutant proteins were by on by AL1 B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S.B. B. Hanley-Bowdoin L. J. PubMed Google Scholar). DNA were as B.M. Hanley-Bowdoin L. J. Google Scholar). pair the AL1 DNA binding A was from and and The DNA was with proteins for 1 the in the The and were on and analyzed by DNA cleavage and to in the of the TGMV origin were 5′ and The and were used for with mutant and AL1 with truncated AL1 only one was and the was the as in the of DNA was with of protein in of cleavage and of for The were by of and to for The were on and analyzed by were and to (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 of DNA a of TGMV B in E.P.B. Gladfelter H.J. Hanley-Bowdoin L. Plant Cell. 1994; 6: PubMed Scopus Google Scholar), mutant AL1 and plant in 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). DNA was and analyzed for double-stranded viral DNA by DNA (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 of are origin recognition and of a for plus-strand DNA TGMV AL1 both of these processes by binding double-stranded DNA in a and by a in the Earlier established that the AL1 N terminus is necessary for both activities B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). region of AL1 conserved sequence and structural motifs a highly predicted pair of α-helices between amino acids and B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of both are conserved among dicot-infecting and the is in that the predicted α-helices necessary for AL1 this we generated site-directed of TGMV AL1 that are in 1 1 and their activities to the protein in vivo and in 1 The were for the to replication of TGMV B DNA in tobacco with TGMV also in a and the AL1 protein of the geminivirus of TGMV B replication 1 and In contrast, has conserved charged residues in to viral DNA replication The mutations in converted conserved charged residues to a the to AL1 mutations replication The mutant established in the amino acid of the predicted are essential for AL1 in The negative of the in that the is also required for AL1 The AL1 in replication were as proteins in by glutathione and for AL1 vitro. was in these experiments of its replication The mutant proteins were for DNA binding in a double-stranded DNA that includes the TGMV AL1 DNA binding of the DNA AL1 the in a The mutant AL1 proteins were also for DNA cleavage a single-stranded to the and of the in the plus-strand the to a and DNA cleavage and These results demonstrated that the predicted 1 and are required both for DNA binding and cleavage by TGMV 1 and are of the AL1 and their have on oligomerization the proteins are and that the of the AL1 to and DNA was to the proteins were for their to form with and of and were with AL1 in as by of protein of the mutant proteins with AL1 on that the mutations AL1/AL1 the DNA binding and cleavage activities of The N terminus of AL1 also includes conserved sequence motifs that are found in initiator proteins Refs. J. PubMed Scopus Google Scholar and Nucleic Acids Res. PubMed Scopus Google Scholar). J. S. Gronenborn B. PubMed Scopus Google Scholar) showed that to the the roles of motifs 1 and in have been We these motifs in TGMV AL1 and analyzed the impact of the mutations on protein function 1 In residues were with In and residues were to and The mutant for the replication that of the 1 TGMV B in tobacco that both motifs are essential for AL1 in the of the mutant replication we proteins to the 1 and from cells and for DNA binding and cleavage in vitro. of the mutant proteins to double-stranded DNA of the single-stranded DNA cleavage In and the DNA experiments established that and and and form with AL1 are Together, these results showed that motifs 1 and of TGMV AL1 are required for both DNA binding and cleavage of includes conserved amino acids that contribute to AL1 We generated TGMV AL1 that one residues in and analyzed their activities in vivo and in vitro. In were for the and conserved TGMV B replication in tobacco and was for DNA cleavage with that double-stranded DNA binding was also a of protein readily the In of DNA binding were with the protein experiments that with AL1 and that the mutant is The DNA binding of was of the of in of DNA cleavage. the of and in DNA we generated two mutations this In was to a viral DNA replication and DNA and that is necessary for these However, only was for DNA binding a of protein DNA binding These results demonstrated that to both the DNA binding and cleavage activities of TGMV These also established that the of is essential for DNA the is required for DNA cleavage. AL1 were to the of conserved charged residues in a of TGMV B replication with the replication DNA binding and DNA cleavage and ligation the mutant of was as well as was to the In and were to a Both to DNA replication and DNA and cleavage and the of the conserved and one acid in AL1 However, results the of and the that the acids in are In H.J. Eagle P.A. E.P.B. Hanley-Bowdoin L. Virology. PubMed Scopus (47) Google Scholar, B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we showed that AL1 amino acids virus-specific recognition of the AL1 binding in amino acids are required for DNA binding vitro. these we that AL1 binds DNA as and that DNA binding two the DNA binding domain in the AL1 N terminus and the oligomerization domain between amino acids and experiments M. Orozco and L. the N-terminal of the AL1 oligomerization domain to the of the AL1 DNA binding we and in cells and the proteins by binding to by with glutathione 1 and The DNA binding of the proteins were in a of protein showed DNA binding DNA protein These established that between amino acids and 130 are required for AL1 DNA binding and that amino acids to the AL1 oligomerization contribute with the The of to DNA a for oligomerization in binding forms B. Crit. Rev. PubMed Scopus Google Scholar). the of oligomerization in was to and with to showed that was in the 1 and DNA binding in The of to form to of the truncated protein was in DNA cleavage these results established that binding is dependent on oligomerization through the amino acids 134–181) a heterologous protein the of the is the of to DNA that dimerization is sufficient for interactions. two cleavage and ligation to form a circular DNA the in vivo we the cleavage and ligation to two A and that in cleavage A was with two to the and 5′ cleavage were A was B was the that both been and that the 5′ of B been to the of A. In B and a cleavage and ligation were the 5′ of B the 5′ of A. was that the ligation of also with the of the 5′ F. S. J. Schaefer S. Schell J. Gronenborn B. Nucleic Acids Res. PubMed Scopus Google Scholar). We then AL1 oligomerization is required for the cleavage and ligation was the as for that the protein was of and with the two and for DNA cleavage and ligation. In and the cleavage and ligation were readily in binding DNA cleavage and ligation are dependent on AL1 Geminiviruses their DNA genomes by a rolling circle to and in (reviewed in Refs. A. DNA and Scholar and G. M. M. 1993; PubMed Scopus Google Scholar) and the in cells Cell. Full Text PDF PubMed Scopus Google Scholar, D.M. J. 1994; PubMed Google Scholar). of these encodes initiator protein that binds double-stranded DNA and catalyzes DNA cleavage and ligation single-stranded DNA The DNA binding recruits the initiator protein to the origin and origin The initiator protein then the plus-strand DNA a to a that is used to DNA synthesis from the of a of a cleavage and ligation a single-stranded of the genome and a double-stranded In geminivirus viral AL1 proteins are the initiator In experiments established other initiator binds double-stranded DNA and catalyzes single-stranded DNA cleavage and ligation in a (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, J. F. Matzeit V. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, B.M. Hanley-Bowdoin L. J. Google Scholar). Both activities have been to the N terminus of F. S. J. Schaefer S. Schell J. Gronenborn B. Nucleic Acids Res. PubMed Scopus Google Scholar, B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and was that conserved structural and sequence motifs in this region are in DNA binding and cleavage. In this we showed that these conserved motifs contribute to DNA binding and cleavage with both activities nearly identical amino However, DNA binding was distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions. The N terminus of TGMV AL1 two of predicted α-helices that are conserved among the proteins of dicot-infecting geminiviruses B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of designated as 1 and are located between amino acids and with amino acids in was that mutations in both geminivirus replication and However, the mutations also DNA that the predicted are also required for cleavage The and for conserved charged residues in showed that the the two is essential for DNA binding and cleavage. the negative of the that that the predicted is required for both activities. the AL1 sequence the of the TGMV AL1 protein in mutant also DNA binding and cleavage. TGMV and AL1 DNA in their plus-strand (14Fontes E.P.B. Gladfelter H.J. Hanley-Bowdoin L. Plant Cell. 1994; 6: PubMed Scopus Google Scholar), is that DNA cleavage is a virus-specific The origin is to is highly conserved among dicot-infecting geminiviruses J. Virology. PubMed Scopus Google Scholar) and between TGMV and (14Fontes E.P.B. Gladfelter H.J. Hanley-Bowdoin L. Plant Cell. 1994; 6: PubMed Scopus Google Scholar). However, showed that N-terminal domains of TGMV and AL1 are in their for recognition and cleavage of their The N terminus of TGMV AL1 also conserved amino acid motifs that are found in geminivirus replication proteins and in initiator proteins Refs. J. PubMed Scopus Google Scholar and Nucleic Acids Res. PubMed Scopus Google Scholar). 1 has of the amino acids of TGMV AL1 that 1 is required for binding and cleavage B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we used site-directed to 1 in the of TGMV that for residues and TGMV B replication in vivo and AL1 DNA binding and cleavage activities in vitro. These results established the of 1 for TGMV AL1 its of the of is that AL1 binds the the and the that binding necessary for origin The sequence is with amino acids 1 on to was that the two in binding necessary for the of initiator proteins Nucleic Acids Res. PubMed Scopus Google Scholar). is with the that is required for DNA cleavage by the protein J. F. Matzeit V. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the in TGMV AL1 DNA replication in vivo and DNA cleavage and binding in is essential for AL1 However, with the and mutant proteins are necessary to is a to the for DNA cleavage J. S. Gronenborn B. PubMed Scopus Google Scholar), a that is conserved in geminivirus The of this for TGMV AL1 function was demonstrated by Both were for vivo and DNA cleavage in vitro. the also the mutant DNA to with and AL1 proteins established that is necessary for replication of these geminiviruses J. S. Gronenborn B. PubMed Scopus Google Scholar, Plant PubMed Scopus Google Scholar). In is required for DNA cleavage J. S. Gronenborn B. PubMed Scopus Google Scholar) and was to in by experiments that is to the 5′ of the DNA cleavage J. S. Gronenborn B. 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In contrast, the on TGMV AL1 function in vivo in vitro. of this is that the acids and for the is with the of a TGMV AL1 mutant for acids and to replication to and the that is essential for TGMV AL1 we that this is for two of AL1 impact on viral DNA replication Plant PubMed Scopus Google Scholar), that this is required for geminivirus proteins most have acid and one both that two residues in this region sufficient for Together, results established the of charged residues in two cleavage and ligation of the DNA the a that to DNA J. S. Gronenborn B. PubMed Scopus Google Scholar), is the cleavage and ligation are geminivirus of the mutations amino acids that are for the cleavage and ligation between the DNA and charged residues in the AL1 is that the residues in AL1 of a are required to both cleavage and ligation. The were to the in vivo by two cleavage to ligation. the oligomerization domain and the through the heterologous protein B. Crit. Rev. PubMed Scopus Google Scholar), both and DNA in these These results demonstrated that AL1 dimerization is required for DNA cleavage and ligation and that AL1 a However, and structural are necessary to the amino in these DNA cleavage and AL1 DNA binding is dependent on AL1/AL1 interactions. is by the that the region of AL1 required for DNA binding overlaps the oligomerization domain B.M. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for the of protein in binding was by a heterologous dimerization domain to a truncated AL1 the oligomerization The protein, through the DNA, the binding also to DNA, that the AL1 DNA binding Together, these results located the AL1 DNA binding domain between experiments TGMV and AL1 proteins virus-specific origin recognition to the N-terminal amino The of amino acids for DNA binding was distinguished with proteins the sequence in this region is identical for TGMV and on is that the DNA binding domains of proteins from and beet curly top virus also the domains for virus-specific origin recognition D.C. Virology. PubMed Scopus Google Scholar, F. B. Gronenborn B. PubMed Scopus Google Scholar). established that the conserved α-helices and cleavage motifs in the AL1 N terminus are required for both DNA binding and However, is that of the mutations that DNA also DNA that is in the amino acids to these is that N-terminal domain of the mutant proteins is for mutations that However, of that for the of of the AL1 most of the mutations were as are one in 1 and in in that TGMV AL1 the conserved mutations in the two and the cleavage motifs AL1 that their were for DNA binding and cleavage. is that the between the amino acid for DNA binding and cleavage essential for DNA binding in the in the for cleavage. the DNA of the AL1 binding and cleavage are the amino acid that these DNA The between DNA binding and cleavage through structural of and mutant AL1 We Settlage and for and reading of the