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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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Related in: MedlinePlus

Activity of AlkB proteins on various [3H]methylated RNA and DNA substrates. (A) [3H]methylated oligonucleotides were incubated with varying amounts of EcAlkB, GVA-36, BlScV-94 or BVY-94 and the ethanol soluble radioactivity released was measured by scintillation counting. (B) [3H]methylated poly(A) was incubated with 100 pmol of BVY94, BVY-94-H59A or BVY-94-D61A. Fe2+ and 2OG were present as indicated. Error bars represent the standard deviation of duplicate measurements.
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Figure 6: Activity of AlkB proteins on various [3H]methylated RNA and DNA substrates. (A) [3H]methylated oligonucleotides were incubated with varying amounts of EcAlkB, GVA-36, BlScV-94 or BVY-94 and the ethanol soluble radioactivity released was measured by scintillation counting. (B) [3H]methylated poly(A) was incubated with 100 pmol of BVY94, BVY-94-H59A or BVY-94-D61A. Fe2+ and 2OG were present as indicated. Error bars represent the standard deviation of duplicate measurements.

Mentions: When ss nucleic acids are treated with methylating agents such as MMS or N-methyl-N-nitrosourea (MNU), substantial amounts of the AlkB substrates 1-meA and, to a lesser extent, 3-meC are introduced (1). To investigate the in vitro repair activity and the substrate specificity of viral AlkB, recombinant GVA-36, BVY-94 and BlScV-94 were incubated with RNA or DNA oligonucleotides that had been [3H]methylated through treatment with [3H]MNU. When comparing the activity on ssRNA relative to ssDNA, EcAlkB was approximately 10-fold more active on ssDNA (Figure 6A), confirming previous observations (14). In contrast, the activity of the viral AlkB proteins was generally higher (up to 10-fold) on ssRNA than on ssDNA (Figure 6A).Figure 6.


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)

Activity of AlkB proteins on various [3H]methylated RNA and DNA substrates. (A) [3H]methylated oligonucleotides were incubated with varying amounts of EcAlkB, GVA-36, BlScV-94 or BVY-94 and the ethanol soluble radioactivity released was measured by scintillation counting. (B) [3H]methylated poly(A) was incubated with 100 pmol of BVY94, BVY-94-H59A or BVY-94-D61A. Fe2+ and 2OG 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 6: Activity of AlkB proteins on various [3H]methylated RNA and DNA substrates. (A) [3H]methylated oligonucleotides were incubated with varying amounts of EcAlkB, GVA-36, BlScV-94 or BVY-94 and the ethanol soluble radioactivity released was measured by scintillation counting. (B) [3H]methylated poly(A) was incubated with 100 pmol of BVY94, BVY-94-H59A or BVY-94-D61A. Fe2+ and 2OG were present as indicated. Error bars represent the standard deviation of duplicate measurements.
Mentions: When ss nucleic acids are treated with methylating agents such as MMS or N-methyl-N-nitrosourea (MNU), substantial amounts of the AlkB substrates 1-meA and, to a lesser extent, 3-meC are introduced (1). To investigate the in vitro repair activity and the substrate specificity of viral AlkB, recombinant GVA-36, BVY-94 and BlScV-94 were incubated with RNA or DNA oligonucleotides that had been [3H]methylated through treatment with [3H]MNU. When comparing the activity on ssRNA relative to ssDNA, EcAlkB was approximately 10-fold more active on ssDNA (Figure 6A), confirming previous observations (14). In contrast, the activity of the viral AlkB proteins was generally higher (up to 10-fold) on ssRNA than on ssDNA (Figure 6A).Figure 6.

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