The presence of protein-nucleic acid covalent bonds is not unusual in viral pathogens. Multiple virus families have a protein covalently attached to their genomes; some examples include Potyviridae, Adenoviridae, Nepoviridae, and Picornaviridae ]. In the case of picornaviruses this viral protein, termed VPg, is located at the 59 end of all nascent picornavirus genomes as a result of serving as a primer for RNA synthesis. The occurrence of protein-nucleic acid covalent bonds in uninfected cells is slightly more novel. These linkages result from the failure of a topoisomerase to dissociate properly from target DNA ]. In such instances additional enzymes, termed tyrosyl-DNA phosphodiesterases, are required to resolve the phosphodiester bond SCH772984 formed with the nucleic acid. Currently, there are two documented examples of these enzymes in eukaryotic cells: tyrosyl-DNA phosphodiesterase 1 and tyrosyl- DNA phosphodiesterase 2, which primarily recognize and cleave 39 and 59 tyrosyl-DNA phosphodiester bonds, respectively. Interestingly, there is evidence for a tyrosyl-RNA phosphodiesterase activity in eukaryotic cells. Existence of this R428 enzyme is demonstrated by the efficient removal of the picornavirus protein VPg, which is attached to the genomic RNA via an O4- tyrosine bond, from the 59 end of the viral genome. Although this activity has been given several names in the literature, including unlinking enzyme, VPg unlinkase, and uridylylpolynucleotide- – tyrosine phosphodiesterase, in this manuscript we will refer to the enzymatic activity as ����unlinkase.���� The identity of unlinkase is unknown, but the activity has been partially characterized. Activity has been reported in wheat germ extracts, mouse ascites Krebs II cells, rabbit reticulocyte lysate, and in the nucleus and cytoplasm of HeLa cells. Unlinkase has the hallmarks of a bonafide enzyme, since the activity is dependent on Mg2+ or Mn2+ and is inhibited in the presence of vanadate, SDS, Zn2+ and EDTA. Reducing agents, translation inhibitors, RNase, and protease inhibitors do not appear to affect unlinkase activity. It is unknown whether unlinkase activity results from a single enzyme, a complex of hetero- or homo-multimers, or an RNP complex with the viral RNA. Partially purified preparations of the enzyme have yielded low turnover numbers, suggesting that the purified protein was only a component of a potential complex or that the enzyme responsible possesses a very different function in the uninfected cell and cleaving of the tyrosyl-RNA bond in picornavirus genomic RNA is simply a minor role of the enzyme. It has also been demonstrated that the length of the attached RNA, not the integrity of the VPg, is more important for efficient unlinkase cleavage.