title: Characterization of polynucleotide kinase/phosphatase enzymes from Mycobacteriophages omega and Cjw1 and vibriophage KVP40.
abstract: Coliphage T4 Pnkp is a bifunctional polynucleotide 5'-kinase/3'-phosphatase that catalyzes the end-healing steps of a RNA repair pathway. Here we show that mycobacteriophages Omega and Cjw1 and vibriophage KVP40 also encode bifunctional Pnkp enzymes consisting of a proximal 5'-kinase module with an essential P-loop motif, GXGK(S/T), and a distal 3'-phosphatase module with an essential acyl-phosphatase motif, DX- DGT. Biochemical characterization of the viral Pnkp proteins reveals several shared features, including an alkaline pH optimum for the kinase component, an intrinsic RNA kinase activity, and a homotetrameric or homodimeric quaternary structure, that distinguish them from the monomeric DNA-specific phosphatase/kinase enzymes found in mammals and fission yeast. Whereas the phage 5'-kinases differ from each other in their preferences for phosphorylation of 5' overhangs, blunt ends, or recessed ends, none of them displays the preference for recessed ends reported for mammalian DNA kinase. We hypothesize that Pnkp provides phages that have it with a means to evade an RNA-damaging antiviral host response. Genetic complementation of the essential end-healing steps of yeast tRNA splicing by the Omega and Cjw1 Pnkp enzymes establishes their capacity to perform RNA repair reactions in vivo. A supportive correlation is that Omega and Cjw1, which are distinguished from other mycobacteriophages by their possession of a Pnkp enzyme, are also unique among the mycobacteriophages in their specification of putative RNA ligases.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhu
authors.forename: Shenmin
authors.initials: S
authors.lastname: Yin
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002412:Cations / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D005816:Genetic Complementation Test; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009160:Mycobacteriophages / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012326:RNA Splicing; D012343:RNA, Transfer / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014733:Vibrio / Q000821:virology*
chemical_list: D002412:Cations; D009841:Oligonucleotides; D011994:Recombinant Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D012343:RNA, Transfer; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010744:Phosphoric Monoester Hydrolases; D005990:Glycerol