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Viral AlkB proteins repair RNA damage by oxidative demethylation.

van den Born E, Omelchenko MV, Bekkelund A, Leihne V, Koonin EV, Dolja VV, Falnes PØ - Nucleic Acids Res. (2008)

Bottom Line: The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro.Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity.Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, N-0316 Oslo, Norway.

ABSTRACT
Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

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Sequence alignment of a part of the nucleotide recognition lid region from various AlkB proteins. The region shown corresponds to aa 63–98 of EcAlkB (gi/113638). Filled arrows indicate residues Trp69 and Tyr76 in EcAlkB, which have been shown to be involved in coordinating 1-meA (30). Dotted arrows indicate residues that are conserved in viral, vertebrate and bacterial AlkB proteins belonging to group 1B, but not to group 1A (29). BlScV, Blueberry scorch virus; BVY, Blackberry virus Y; GVA, Grapevine virus A; LChV-2, Little cherry virus 2; CLBV, Citric leave blotch virus, CVB, Chrysanthemum virus B; LSV, Lily symptomless virus; ASPV, Apple stem pitting virus; GLV, garlic latent virus; ZVX, Zygocactus virus X; Eco, E. coli; Sme, Sinorhizobium meliloti; Rme, Ralstonia metallidurans; Ppu, Pseudomonas putida; Vvu, Vibrio vulnificus; Pae, Pseudomonas aeruginosa; Xca, Xanthomonas campestris; Son, Shewanella oneidensis; Hs, Homo sapiens; Gg, Gallus gallus; Dr, Danio rerio.
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Figure 2: Sequence alignment of a part of the nucleotide recognition lid region from various AlkB proteins. The region shown corresponds to aa 63–98 of EcAlkB (gi/113638). Filled arrows indicate residues Trp69 and Tyr76 in EcAlkB, which have been shown to be involved in coordinating 1-meA (30). Dotted arrows indicate residues that are conserved in viral, vertebrate and bacterial AlkB proteins belonging to group 1B, but not to group 1A (29). BlScV, Blueberry scorch virus; BVY, Blackberry virus Y; GVA, Grapevine virus A; LChV-2, Little cherry virus 2; CLBV, Citric leave blotch virus, CVB, Chrysanthemum virus B; LSV, Lily symptomless virus; ASPV, Apple stem pitting virus; GLV, garlic latent virus; ZVX, Zygocactus virus X; Eco, E. coli; Sme, Sinorhizobium meliloti; Rme, Ralstonia metallidurans; Ppu, Pseudomonas putida; Vvu, Vibrio vulnificus; Pae, Pseudomonas aeruginosa; Xca, Xanthomonas campestris; Son, Shewanella oneidensis; Hs, Homo sapiens; Gg, Gallus gallus; Dr, Danio rerio.

Mentions: As discussed in the previous paragraph, viral AlkBs display a high degree of similarity to bacterial AlkB proteins, particularly those belonging to group 1B (29), as well as to ABH2 and ABH3, which represent vertebrate AlkB proteins already reported to be involved in nucleic acid repair (9,14). Interestingly, the viral, vertebrate and bacterial AlkBs shared several conserved amino acid residues in a region corresponding to the EcAlkB nucleotide recognition lid (Figure 2). Two of these residues, Trp69 and Tyr76, were found to be in close contact with the 1-meA substrate in the EcAlkB 3D-structure (30). Therefore, our initial bioinformatics analysis suggested that the viral AlkB proteins were likely to possess a repair activity similar to that previously reported for EcAlkB, ABH2 and ABH3, and experiments were designed to further explore this possibility.Figure 2.


Viral AlkB proteins repair RNA damage by oxidative demethylation.

van den Born E, Omelchenko MV, Bekkelund A, Leihne V, Koonin EV, Dolja VV, Falnes PØ - Nucleic Acids Res. (2008)

Sequence alignment of a part of the nucleotide recognition lid region from various AlkB proteins. The region shown corresponds to aa 63–98 of EcAlkB (gi/113638). Filled arrows indicate residues Trp69 and Tyr76 in EcAlkB, which have been shown to be involved in coordinating 1-meA (30). Dotted arrows indicate residues that are conserved in viral, vertebrate and bacterial AlkB proteins belonging to group 1B, but not to group 1A (29). BlScV, Blueberry scorch virus; BVY, Blackberry virus Y; GVA, Grapevine virus A; LChV-2, Little cherry virus 2; CLBV, Citric leave blotch virus, CVB, Chrysanthemum virus B; LSV, Lily symptomless virus; ASPV, Apple stem pitting virus; GLV, garlic latent virus; ZVX, Zygocactus virus X; Eco, E. coli; Sme, Sinorhizobium meliloti; Rme, Ralstonia metallidurans; Ppu, Pseudomonas putida; Vvu, Vibrio vulnificus; Pae, Pseudomonas aeruginosa; Xca, Xanthomonas campestris; Son, Shewanella oneidensis; Hs, Homo sapiens; Gg, Gallus gallus; Dr, Danio rerio.
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Related In: Results  -  Collection

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Figure 2: Sequence alignment of a part of the nucleotide recognition lid region from various AlkB proteins. The region shown corresponds to aa 63–98 of EcAlkB (gi/113638). Filled arrows indicate residues Trp69 and Tyr76 in EcAlkB, which have been shown to be involved in coordinating 1-meA (30). Dotted arrows indicate residues that are conserved in viral, vertebrate and bacterial AlkB proteins belonging to group 1B, but not to group 1A (29). BlScV, Blueberry scorch virus; BVY, Blackberry virus Y; GVA, Grapevine virus A; LChV-2, Little cherry virus 2; CLBV, Citric leave blotch virus, CVB, Chrysanthemum virus B; LSV, Lily symptomless virus; ASPV, Apple stem pitting virus; GLV, garlic latent virus; ZVX, Zygocactus virus X; Eco, E. coli; Sme, Sinorhizobium meliloti; Rme, Ralstonia metallidurans; Ppu, Pseudomonas putida; Vvu, Vibrio vulnificus; Pae, Pseudomonas aeruginosa; Xca, Xanthomonas campestris; Son, Shewanella oneidensis; Hs, Homo sapiens; Gg, Gallus gallus; Dr, Danio rerio.
Mentions: As discussed in the previous paragraph, viral AlkBs display a high degree of similarity to bacterial AlkB proteins, particularly those belonging to group 1B (29), as well as to ABH2 and ABH3, which represent vertebrate AlkB proteins already reported to be involved in nucleic acid repair (9,14). Interestingly, the viral, vertebrate and bacterial AlkBs shared several conserved amino acid residues in a region corresponding to the EcAlkB nucleotide recognition lid (Figure 2). Two of these residues, Trp69 and Tyr76, were found to be in close contact with the 1-meA substrate in the EcAlkB 3D-structure (30). Therefore, our initial bioinformatics analysis suggested that the viral AlkB proteins were likely to possess a repair activity similar to that previously reported for EcAlkB, ABH2 and ABH3, and experiments were designed to further explore this possibility.Figure 2.

Bottom Line: The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro.Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity.Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, N-0316 Oslo, Norway.

ABSTRACT
Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

Show MeSH
Related in: MedlinePlus