title: Enzyme assays for synthesis and degradation of 2-5As and other 2'-5' oligonucleotides.
abstract: BACKGROUND
  The 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.
  
  
  RESULTS
  Here we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2'-5' oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD(+) as substrates is also presented. The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2'-5' oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays.
  
  
  CONCLUSIONS
  This paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, DK-8000, Aarhus C, Denmark. JESP@ssi.dk.
authors.forename: Jesper Buchhave
authors.initials: JB
authors.lastname: Poulsen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, DK-8000, Aarhus C, Denmark. karina.hansen.kjer@dupont.com.
authors.forename: Karina Hansen
authors.initials: KH
authors.lastname: Kjær
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, DK-8000, Aarhus C, Denmark. jj@mbg.au.dk.
authors.forename: Just
authors.initials: J
authors.lastname: Justesen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, DK-8000, Aarhus C, Denmark. pmm@mbg.au.dk.
authors.forename: Pia Møller
authors.initials: PM
authors.lastname: Martensen
mesh_terms: D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism; D000227:Adenine Nucleotides / Q000096:biosynthesis* / Q000378:metabolism*; D057075:Enzyme Assays; D005095:Exoribonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009243:NAD / Q000378:metabolism; D009843:Oligoribonucleotides / Q000096:biosynthesis* / Q000378:metabolism*; D011131:Polyribonucleotides / Q000096:biosynthesis* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; D011131:Polyribonucleotides; D009243:NAD; C023505:2',5'-oligoadenylate; C475085:OAS1 protein, human; D015088:2',5'-Oligoadenylate Synthetase; D005095:Exoribonucleases; C568404:PDE12 protein, human