Tpt1 catalyzes the transfer of an internal 2'-monophosphate moiety (2'-PO) from a "branched" 2'-PO RNA splice junction to NAD to form a "clean" 2'-OH, 3'-5' phosphodiester junction, ADP-ribose 1″-2″ cyclic phosphate, and nicotinamide. First discovered as an essential component of the tRNA splicing machinery, Tpt1 is widely distributed in nature, including in taxa that have no yeast-like RNA splicing system. Here we characterize the RslTpt1 protein from the bacterium, in which Tpt1 is encoded within a putative RNA repair gene cluster. We find that (i) expression of RslTpt1 in yeast complements a lethal Δ knockout, and (ii) purified recombinant RslTpt1 is a bona fide NAD-dependent 2'-phosphotransferase capable of completely removing an internal 2'-phosphate from synthetic RNAs. The in vivo activity of RslTpt1 is abolished by alanine substitutions for conserved amino acids Arg16, His17, Arg64, and Arg119. The R64A, R119A, and H17A mutants accumulate high levels of a 2'-phospho-ADP-ribosylated RNA reaction intermediate (2'-P-ADPR, evanescent in the wild-type RslTpt1 reaction), which is converted slowly to a 2'-OH RNA product. The R16A mutant is 300-fold slower than wild-type RslTpt1 in forming the 2'-P-ADPR intermediate. Whereas wild-type RsTpt1 rapidly converts the isolated 2'-P-ADPR intermediate to 2'-OH product in the absence of NAD, the H17A, R119A, R64A, and R16A mutant are slower by factors of 3, 33, 210, and 710, respectively. Our results identify active site constituents involved in the catalysis of step 1 and step 2 of the Tpt1 reaction pathway.

RNA 2′-phosphotransferase Tpt1 converts an internal RNA 2′-monophosphate to a 2′-OH via a two-step NAD+-dependent mechanism in which: (i) the 2′-phosphate attacks the C1″ of NAD+ to expel nicotinamide and form a 2′-phospho-ADP-ribosylated RNA intermediate; and (ii) the ADP-ribose O2″ attacks the phosphate of the RNA 2′-phospho-ADPR intermediate to expel the RNA 2′-OH and generate ADP-ribose 1″–2″ cyclic phosphate. Tpt1 is an essential component of the fungal tRNA splicing pathway that generates a unique 2′-PO4, 3′-5′ phosphodiester splice junction during tRNA ligation. The wide distribution of Tpt1 enzymes in taxa that have no fungal-type RNA ligase raises the prospect that Tpt1 might catalyze reactions other than RNA 2′-phosphate removal. A survey of Tpt1 enzymes from diverse sources reveals that whereas all of the Tpt1 enzymes are capable of NAD+-dependent conversion of an internal RNA 2′-PO4 to a 2′-OH (the canonical Tpt1 reaction), a subset of Tpt1 enzymes also catalyzed NAD+-dependent ADP-ribosylation of an RNA or DNA 5′-monophosphate terminus. Aeropyrum pernix Tpt1 (ApeTpt1) is particularly adept in this respect. One-step synthesis of a 5′-phospho-ADP-ribosylated cap structure by ApeTpt1 (with no subsequent 5′-phosphotransferase step) extends the repertoire of the Tpt1 enzyme family and the catalogue of ADP-ribosylation reactions involving nucleic acid acceptors.

Tpt1 is an essential agent of fungal tRNA splicing that removes the 2′-PO4 at the splice junction generated by fungal tRNA ligase. Tpt1 catalyzes a unique two-step reaction whereby the 2′-PO4 attacks NAD+ to form an RNA-2′-phospho-ADP-ribosyl intermediate that undergoes transesterification to yield 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. Because Tpt1 is inessential in exemplary bacterial and mammalian taxa, Tpt1 is seen as an attractive antifungal target. Here we report a 1.4 Å crystal structure of Tpt1 in a product-mimetic complex with ADP-ribose-1″-phosphate in the NAD+ site and pAp in the RNA site. The structure reveals how Tpt1 recognizes a 2′-PO4 RNA splice junction and the mechanism of RNA phospho-ADP-ribosylation. This study also provides evidence that a bacterium has an endogenous phosphorylated substrate with which Tpt1 reacts.

5=-and 3=-end-healing reactions are key steps in nucleic acid break repair in which 5=-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3=-PO 4 or 2=,3=-cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5=-PO 4 and 3=-OH termini required for sealing by classic polynucleotide ligases. Endhealing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2=,3=-phosphoesterase HD domain and a C-terminal 5=-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5=-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2=,3=-cyclic phosphate, RNA 3=-phosphate, RNA 2=-phosphate, and DNA 3=-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCEThe present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5=-and 3=-end-healing enzymes that phosphorylate 5=-OH termini and dephosphorylate 2=,3=-cyclic-PO 4 , 3=-PO 4 , and 2=-PO 4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla. KEYWORDS 3= phosphatase, nucleic acid repair, polynucleotide kinase P olynucleotide kinases (Pnks) are a widely distributed class of cellular and virusencoded nucleic acid repair enzymes that convert 5=-OH termini into 5=-PO 4 ends that can be sealed by RNA or DNA ligases. Pnks are members of the P-loop phosphotransferase superfamily; they catalyze metal-dependent transfer of the ␥ phosphate of a nucleoside triphosphate (NTP) donor to a 5=-OH polynucleotide acceptor. In many repair systems, a Pnk enzyme is fused in a modular fashion to one or more other repair enzymes within a single multifunctional polypeptide.For example, bacteriophage T4 encodes a bifunctional 5=-OH polynucleotide kinase-3=-phosphatase (Pnkp) consisting of an N-terminal Pnk domain fused to a C-terminal

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