There are many biological contexts in which DNA damage generates "dirty" breaks with 3′-PO 4 (or cyclic-PO 4 ) and 5′-OH ends that cannot be sealed by DNA ligases. Here we show that the Escherichia coli RNA ligase RtcB can splice these dirty DNA ends via a unique chemical mechanism. RtcB transfers GMP from a covalent RtcB-GMP intermediate to a DNA 3′-PO 4 to form a "capped" 3′ end structure, DNA 3′ pp 5′ G. When a suitable DNA 5′-OH end is available, RtcB catalyzes attack of the 5′-OH on DNA 3′ pp 5′ G to form a 3′-5′ phosphodiester splice junction. Our findings unveil an enzymatic capacity for DNA 3′ capping and the sealing of DNA breaks with 3′-PO 4 and 5′-OH termini, with implications for DNA repair and DNA rearrangements.T he Escherichia coli RtcB is a founding member of a recently discovered family of RNA repair/splicing enzymes that join RNA 2′,3′-cyclic-PO 4 or 3′-PO 4 ends to RNA 5′-OH ends (1-4). RtcB executes a four-step pathway that requires GTP as an energy source and Mn 2+ as a cofactor (5-7). RtcB first reacts with GTP to form a covalent RtcB-(histidinyl 337 -N)-GMP intermediate. It then hydrolyzes the RNA 2′,3′-cyclic-PO 4 end to a 3′-PO 4 and transfers guanylate from His337 to the RNA 3′-PO 4 to form an RNA 3′ pp 5′ G intermediate. Finally, RtcB catalyzes the attack of an RNA 5′-OH on the RNA 3′ pp 5′ G end to form the 3′-5′ phosphodiester splice junction and liberate GMP.The unique chemical mechanism of RtcB overturned a longstanding tenet of nucleic acid enzymology, which held that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′-phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A 5′ pp 5′ N, in the case of classic RNA/DNA ligases. In light of the wide distribution of RtcB proteins in bacteria, archaea, and metazoa, we raised the prospect of an alternative enzymology based on covalently activated 3′-PO 4 ends (6).In principle, the chemistry of RNA 3′-PO 4 /5′-OH end joining by RtcB might be portable to DNA transactions and pertinent to DNA repair. A variety of hydrolytic nucleases incise the DNA phosphodiester backbone to yield 3′-PO 4 and 5′-OH termini that cannot be joined by DNA ligases. Nonligatable 3′-PO 4 ends are also generated during base excision repair catalyzed by DNA glycosylase/lyase enzymes, during the repair of trapped covalent topoisomerase IB-DNA adducts by tyrosyl-DNA phosphodiesterase 1, and during DNA damage inflicted by ionizing radiation. One way nature solves this "dirty end" problem is by deploying a variety of "end healing" enzymes (8-14). These include 3′-phosphoesterases that convert a 3′-PO 4 to a 3′-OH and 5′-kinases that transform a 5′-OH to a 5′-PO 4 , thereby enabling break sealing by the classic ligase pathway. Given what we now know about RtcB, would it not make sense for nature to also endow a pathway for direct joining of DNA 3′-PO 4 and 5′-OH ends, be it via RtcB or another ligase yet to be discovered?We can extend this thought to DN...

tRNA ligases are essential components of informational and stress-response pathways entailing repair of RNA breaks with 2 ′ ,3 ′ -cyclic phosphate and 5 ′ -OH ends. Plant and fungal tRNA ligases comprise three catalytic domains. Phosphodiesterase and kinase modules heal the broken ends to generate the 3 ′ -OH, 2 ′ -PO 4 , and 5 ′ -PO 4 required for sealing by the ligase. We exploit RNA substrates with different termini to define rates of individual steps or subsets of steps along the repair pathway of plant ligase AtRNL. The results highlight rate-limiting transactions, how repair is affected by active-site mutations, and how mutations are bypassed by RNA alterations. We gain insights to 2 ′ -PO 4 specificity by showing that AtRNL is deficient in transferring AMP to pRNA OH to form AppRNA OH but proficient at sealing pre-adenylylated AppRNA OH . This strategy for discriminating 2 ′ -PO 4 versus 2 ′ -OH ends provides a quality-control checkpoint to ensure that only purposeful RNA breaks are sealed and to avoid nonspecific "capping" of 5 ′ -PO 4 ends.

2H (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse RNA and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. Here we report a biochemical and structural characterization of the Escherichia coli 2H protein YadP, which was identified originally as a reversible transesterifying "nuclease/ligase" at RNA 2 ′ ,5 ′ -phosphodiesters. We find that YadP is an "end healing" cyclic phosphodiesterase (CPDase) enzyme that hydrolyzes an HO RNA>p substrate with a 2 ′ ,3 ′ -cyclic phosphodiester to a HO RNAp product with a 2 ′ -phosphomonoester terminus, without concomitant end joining. Thus we rename this enzyme ThpR (two-histidine 2 ′ ,3 ′ -cyclic phosphodiesterase acting on RNA). The 2.0 Å crystal structure of ThpR in a product complex with 2 ′ -AMP highlights the roles of extended histidine-containing motifs 43 ′ leaving group, thereby implicating His43 as a general acid catalyst. His125-Nε coordinates the O1P oxygen of the AMP 2 ′ -phosphate (inferred from geometry to derive from the attacking water nucleophile), pointing to His125 as a general base catalyst. Arg130 makes bidentate contact with the AMP 2 ′ -phosphate, suggesting a role in transition-state stabilization. Consistent with these inferences, changing His43, His125, or Arg130 to alanine effaced the CPDase activity of ThpR. Phe48 makes a π-π stack on the adenine nucleobase. Mutating Phe28 to alanine slowed the CPDase by an order of magnitude. The tertiary structure and extended active site motifs of ThpR are conserved in a subfamily of bacterial and archaeal 2H enzymes.

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that repairs RNA breaks with 2 ′ ,3 ′ -cyclic-PO 4 and 5 ′ -OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3 ′ -OH, 2 ′ -PO 4 , and 5 ′ -PO 4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and they are promising targets for antifungal drug discovery because: (i) their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme; and (ii) there are no obvious homologs of the Trl1 ligase domain in mammalian proteomes. Here we characterize the tRNA ligases of two human fungal pathogens: Coccidioides immitis and Aspergillus fumigatus. The biological activity of CimTrl1 and AfuTrl1 was verified by showing that their expression complements a Saccharomyces cerevisiae trl1Δ mutant. Purified recombinant AfuTrl1 and CimTrl1 proteins were catalytically active in joining 2 ′ ,3 ′ -cyclic-PO 4 and 5 ′ -OH ends in vitro, either as full-length proteins or as a mixture of separately produced healing and sealing domains. The biochemical properties of CimTrl1 and AfuTrl1 are similar to those of budding yeast Trl1, particularly with respect to their preferential use of GTP as the phosphate donor for the polynucleotide kinase reaction. Our findings provide genetic and biochemical tools to screen for inhibitors of tRNA ligases from pathogenic fungi.

Plant and fungal tRNA ligases are trifunctional enzymes that repair RNA breaks with 2 ′ ,3 ′ -cyclic-PO 4 and 5 ′ -OH ends. They are composed of cyclic phosphodiesterase (CPDase) and polynucleotide kinase domains that heal the broken ends to generate the 3 ′ -OH, 2 ′ -PO 4 , and 5 ′ -PO 4 required for sealing by a ligase domain. Here, we use short HO RNA>p substrates to determine, in a one-pot assay format under single-turnover conditions, the order and rates of the CPDase, kinase and ligase steps. The observed reaction sequence for the plant tRNA ligase AtRNL, independent of RNA length, is that the CPDase engages first, converting HO RNA>p to HO RNA 2 ′ p, which is then phosphorylated to pRNA 2 ′ p by the kinase. Whereas the rates of the AtRNL CPDase and kinase reactions are insensitive to RNA length, the rate of the ligase reaction is slowed by a factor of 16 in the transition from 10-mer RNA to 8-mer and further by eightfold in the transition from 8-mer RNA to 6-mer. We report that a single ribonucleoside-2 ′ ,3 ′ -cyclic-PO 4 moiety enables AtRNL to efficiently splice an otherwise all-DNA strand. Our characterization of a fungal tRNA ligase (KlaTrl1) highlights important functional distinctions vis à vis the plant homolog. We find that (1) the KlaTrl1 kinase is 300-fold faster than the AtRNL kinase; and (2) the KlaTrl1 kinase is highly specific for GTP or dGTP as the phosphate donor. Our findings recommend tRNA ligase as a tool to map ribonucleotides embedded in DNA and as a target for antifungal drug discovery.