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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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AlkB-mediated decarboxylation of 2-oxoglutarate. (A) Purified recombinant His-tagged AlkB proteins used in this study. Proteins were visualized by coomassie staining of a 15% SDS–PAGE gel. (B) Reaction mixtures containing [5-14C]2-oxoglutarate, 100 pmol of EcAlkB and 500 pmol of GVA-36, BlScV-94, BVY-94, BVY-94-H59A or BVY-94-D61A were incubated for 30 min at 37°C, and the released [1-14C]succinate was measured by scintillation counting. Cofactor Fe2+ and substrate-mimic 1-methyladenosine (m1A) were present as indicated. Error bars represent the standard deviation of duplicate measurements.
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Figure 5: AlkB-mediated decarboxylation of 2-oxoglutarate. (A) Purified recombinant His-tagged AlkB proteins used in this study. Proteins were visualized by coomassie staining of a 15% SDS–PAGE gel. (B) Reaction mixtures containing [5-14C]2-oxoglutarate, 100 pmol of EcAlkB and 500 pmol of GVA-36, BlScV-94, BVY-94, BVY-94-H59A or BVY-94-D61A were incubated for 30 min at 37°C, and the released [1-14C]succinate was measured by scintillation counting. Cofactor Fe2+ and substrate-mimic 1-methyladenosine (m1A) were present as indicated. Error bars represent the standard deviation of duplicate measurements.

Mentions: Many Fe(II)/2OG-dependent dioxygenases are able to catalyse the decarboxylation of the cosubstrate 2-oxoglutarate to succinate at a low rate even in the absence of their primary substrate (‘uncoupled reaction’), and this is also the case for EcAlkB (7,8). To confirm that viral AlkBs represent 2OG-dependent enzymes, recombinant GVA-36, BVY-94 and BlScV-94, were expressed in E. coli and purified as hexahistidine-tagged proteins (Figure 5A), and then tested for their ability to carry out the conversion of 2-oxoglutarate to succinate. All three proteins showed robust decarboxylation activity in the absence of any substrate, and the reaction could be stimulated by the substrate-mimic 1-methyladenosine (Figure 5B), which was previously shown to stimulate this reaction in the case of EcAlkB (33). Omitting Fe2+ from the reaction buffer reduced decarboxylation activity drastically (Figure 5B). The mutants BVY-94-H59A and BVY-94-D61A were both unable to convert 2-oxoglutarate to succinate (Figure 5B). Taken together, these results confirmed that the viral AlkBs are iron(II)- and 2-oxoglutarate-dependent repair enzymes, and indicated that these proteins are able to accommodate 1-meA in their active site.Figure 5.


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)

AlkB-mediated decarboxylation of 2-oxoglutarate. (A) Purified recombinant His-tagged AlkB proteins used in this study. Proteins were visualized by coomassie staining of a 15% SDS–PAGE gel. (B) Reaction mixtures containing [5-14C]2-oxoglutarate, 100 pmol of EcAlkB and 500 pmol of GVA-36, BlScV-94, BVY-94, BVY-94-H59A or BVY-94-D61A were incubated for 30 min at 37°C, and the released [1-14C]succinate was measured by scintillation counting. Cofactor Fe2+ and substrate-mimic 1-methyladenosine (m1A) were present as indicated. Error bars represent the standard deviation of duplicate measurements.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553587&req=5

Figure 5: AlkB-mediated decarboxylation of 2-oxoglutarate. (A) Purified recombinant His-tagged AlkB proteins used in this study. Proteins were visualized by coomassie staining of a 15% SDS–PAGE gel. (B) Reaction mixtures containing [5-14C]2-oxoglutarate, 100 pmol of EcAlkB and 500 pmol of GVA-36, BlScV-94, BVY-94, BVY-94-H59A or BVY-94-D61A were incubated for 30 min at 37°C, and the released [1-14C]succinate was measured by scintillation counting. Cofactor Fe2+ and substrate-mimic 1-methyladenosine (m1A) were present as indicated. Error bars represent the standard deviation of duplicate measurements.
Mentions: Many Fe(II)/2OG-dependent dioxygenases are able to catalyse the decarboxylation of the cosubstrate 2-oxoglutarate to succinate at a low rate even in the absence of their primary substrate (‘uncoupled reaction’), and this is also the case for EcAlkB (7,8). To confirm that viral AlkBs represent 2OG-dependent enzymes, recombinant GVA-36, BVY-94 and BlScV-94, were expressed in E. coli and purified as hexahistidine-tagged proteins (Figure 5A), and then tested for their ability to carry out the conversion of 2-oxoglutarate to succinate. All three proteins showed robust decarboxylation activity in the absence of any substrate, and the reaction could be stimulated by the substrate-mimic 1-methyladenosine (Figure 5B), which was previously shown to stimulate this reaction in the case of EcAlkB (33). Omitting Fe2+ from the reaction buffer reduced decarboxylation activity drastically (Figure 5B). The mutants BVY-94-H59A and BVY-94-D61A were both unable to convert 2-oxoglutarate to succinate (Figure 5B). Taken together, these results confirmed that the viral AlkBs are iron(II)- and 2-oxoglutarate-dependent repair enzymes, and indicated that these proteins are able to accommodate 1-meA in their active site.Figure 5.

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