Determination of the origin cleavage and joining domain of geminivirus Rep proteins

Heyraud-Nitschke, Françoise; Schumacher, S.; Laufs, Jürgen; Schaefer, Sabine; Schell, Jeff; Gronenborn, Bruno

doi:10.1093/nar/23.6.910

Cited by 148 publications

(99 citation statements)

References 34 publications

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“…2F). The putative Rep has two conserved domains, namely geminivirus Rep catalytic domain (Gemini_AL1) and geminivirus Rep protein central domain (Gemini_AL1_M) with conserved motifs for rolling-circle replication (21) (Fig. 3A).…”

Section: Resultsmentioning

confidence: 99%

A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus

Fù

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

466

333

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Mycoviruses are viruses that infect fungi and have the potential to control fungal diseases of crops when associated with hypovirulence. Typically, mycoviruses have double-stranded (ds) or singlestranded (ss) RNA genomes. No mycoviruses with DNA genomes have previously been reported. Here, we describe a hypovirulenceassociated circular ssDNA mycovirus from the plant pathogenic fungus Sclerotinia sclerotiorum. The genome of this ssDNA virus, named Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), is 2166 nt, coding for a replication initiation protein (Rep) and a coat protein (CP). Although phylogenetic analysis of Rep showed that SsHADV-1 is related to geminiviruses, it is notably distinct from geminiviruses both in genome organization and particle morphology. Polyethylene glycol-mediated transfection of fungal protoplasts was successful with either purified SsHADV-1 particles or viral DNA isolated directly from infected mycelium. The discovery of an ssDNA mycovirus enhances the potential of exploring fungal viruses as valuable tools for molecular manipulation of fungi and for plant disease control and expands our knowledge of global virus ecology and evolution.

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Section: Resultsmentioning

confidence: 99%

A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus

Fù

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

466

333

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“…hydrogen bonding of the tyrosine to the phosphodiester to be cleaved, was shown by the crystal structure of the Klenow fragment of polymerase I complexed with single-stranded DNA [46]. Also in geminivirus Rep proteins, the same backbone spacing of critical amino acids is conserved (DVKXYXXKD or YXXKD/E/N) which may suggest that in a metal assisted hydrolysis they may coordinate the divalent cation required for cleavage by both TYLCV and WDV Rep protein [26,28], and to correctly position the scissile phosphodiester bond [47].…”

Section: Discussionmentioning

confidence: 99%

“…The presence of a 40 nt/39 nt double band and of the 24 nt/23 nt (lanes 2 and 3) is due to a co-purified exonuclease activity in this particular batch of proteins. The transfer of the single 3" terminal nucleotide of TY39/1 from Rep protein to the 3' acceptor end of the labelled I 1 nt cleavage product occurs with very low efficiency, see also [26]. Hence, the corresponding 12 nt labelled joining product was not detectable.…”

Section: Repylo3f Has Neither Dna Cleavage Nor Nucleotidyl Transfer Amentioning

confidence: 97%

“…Construction of the expressi,m plasmid and purification of the fusion protein was as described [26,28]. 5.8 nmol GST-RepNz4 bound to 0.7 ml glutathione-beads were suspended in a final volume of 6 ml buffer A (25 mM Tris-HCl pH 7.5, 75 mM NaC1, 2.5 mM MgC12, 2.5 mM MnC12, 2.5 mM DTT, 0.5 mM EDTA) containing the oligonucleotide cleavage substrate mix.…”

Section: ~ Formation Of a Covalent Gst-repwo3-amp Adductmentioning

confidence: 99%

“…For this purpose, we used the amino-terminal domain of Rep (GST-RepN24) which had been shown earlier to cleave and ligate the origin of replication as efficiently as the full-size protein [26]. 5.8 nmoles of GST-RepN24 were bound to glutathione-agarose beads, and a cleavage reaction of 23 nmoles substrate oligonucleotide mix, containing 38 pmoles of oligonucleotide TY39/0 labelled at its 3' end (see section 2 for details) was performed with the bound protein.…”

Section: Identification Of the Amino Acid That Landks Rep To Dnamentioning

confidence: 99%

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Identification of the nicking tyrosine of geminivirus Rep protein

et al. 1995

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Two tyrosines, both located in the amino-terminal domain of the protein, are conserved in geminivirus Rep proteins. The first one is part of the sequence motif-FLTY-whose function is still unknown, and the second one is the central amino acid of a sequence that has been suggested to catalyze replication initiation [33] (see Fig. 1) Here we provide biochemical proof that the tyrosine-103 of the Rep protein tomato yellow leaf curl virus (TYLCV) is the active amino acid of the cleavage/joining reaction.

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Geminivirus vectors for high‐level expression of foreign proteins in plant cells

Mor

Moon

Palmer

et al. 2002

Biotech & Bioengineering

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Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.

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Determination of the origin cleavage and joining domain of geminivirus Rep proteins

Cited by 148 publications

References 34 publications

A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus

A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus

Identification of the nicking tyrosine of geminivirus Rep protein

Geminivirus vectors for high‐level expression of foreign proteins in plant cells

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