Limits...
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.

Show MeSH

Related in: MedlinePlus

Reactivation of MMS-treated bacteriophages by expression of AlkB proteins in E. coli. (A) Reactivation of methylated ssRNA phage MS2 by AlkB proteins from GVA, BVY, BlScV, LChV-2 or CLBV. Reactivation of methylated MS2 by different variants of AlkB from (B) GVA, (C) BlScV and (D) BVY. (E) Reactivation of methylated ssDNA phage M13 by GVA-36, BVY-36 and BlScV-94. (F) Reactivation of methylated MS2 by BVY-94 and mutants BVY-94-H59A and BVY-94-D61A. Expression plasmid pJB658 without insert was used as control. Error bars in B–D represent the standard deviation of triplicate measurements.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553587&req=5

Figure 4: Reactivation of MMS-treated bacteriophages by expression of AlkB proteins in E. coli. (A) Reactivation of methylated ssRNA phage MS2 by AlkB proteins from GVA, BVY, BlScV, LChV-2 or CLBV. Reactivation of methylated MS2 by different variants of AlkB from (B) GVA, (C) BlScV and (D) BVY. (E) Reactivation of methylated ssDNA phage M13 by GVA-36, BVY-36 and BlScV-94. (F) Reactivation of methylated MS2 by BVY-94 and mutants BVY-94-H59A and BVY-94-D61A. Expression plasmid pJB658 without insert was used as control. Error bars in B–D represent the standard deviation of triplicate measurements.

Mentions: An AlkB protein active on RNA (e.g. EcAlkB or hABH3) is, when expressed in AlkB-deficient E. coli, able to increase the survival of RNA bacteriophage MS2 inactivated by treatment with a methylating compound (e.g. MMS), due to repair of deleterious methyl lesions in the viral RNA (14). In initial experiments, AlkB proteins derived from the five selected viruses were tested for such repair activity. Robust repair activity, comparable to that of EcAlkB, was observed for GVA-36, BVY-36 and BlScV-94, while only low activity was detected in the case of LChV-2-50 and CLBV-36 (Figure 4A). Based on these results, the viral AlkB proteins from GVA, BVY and BlScV were selected for further analysis.Figure 4.


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)

Reactivation of MMS-treated bacteriophages by expression of AlkB proteins in E. coli. (A) Reactivation of methylated ssRNA phage MS2 by AlkB proteins from GVA, BVY, BlScV, LChV-2 or CLBV. Reactivation of methylated MS2 by different variants of AlkB from (B) GVA, (C) BlScV and (D) BVY. (E) Reactivation of methylated ssDNA phage M13 by GVA-36, BVY-36 and BlScV-94. (F) Reactivation of methylated MS2 by BVY-94 and mutants BVY-94-H59A and BVY-94-D61A. Expression plasmid pJB658 without insert was used as control. Error bars in B–D represent the standard deviation of triplicate measurements.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Reactivation of MMS-treated bacteriophages by expression of AlkB proteins in E. coli. (A) Reactivation of methylated ssRNA phage MS2 by AlkB proteins from GVA, BVY, BlScV, LChV-2 or CLBV. Reactivation of methylated MS2 by different variants of AlkB from (B) GVA, (C) BlScV and (D) BVY. (E) Reactivation of methylated ssDNA phage M13 by GVA-36, BVY-36 and BlScV-94. (F) Reactivation of methylated MS2 by BVY-94 and mutants BVY-94-H59A and BVY-94-D61A. Expression plasmid pJB658 without insert was used as control. Error bars in B–D represent the standard deviation of triplicate measurements.
Mentions: An AlkB protein active on RNA (e.g. EcAlkB or hABH3) is, when expressed in AlkB-deficient E. coli, able to increase the survival of RNA bacteriophage MS2 inactivated by treatment with a methylating compound (e.g. MMS), due to repair of deleterious methyl lesions in the viral RNA (14). In initial experiments, AlkB proteins derived from the five selected viruses were tested for such repair activity. Robust repair activity, comparable to that of EcAlkB, was observed for GVA-36, BVY-36 and BlScV-94, while only low activity was detected in the case of LChV-2-50 and CLBV-36 (Figure 4A). Based on these results, the viral AlkB proteins from GVA, BVY and BlScV were selected for further analysis.Figure 4.

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