Nucleotidyltransferase Superfamily Research Articles

Acquisition of pcnB [poly(A) polymerase I] genes via horizontal transfer from the β, γ-Proteobacteria

Poly(A) polymerases (PAPs) and tRNA nucleotidyltransferases belong to a superfamily of nucleotidyltransferases and modify RNA 3′-ends. The product of the pcnB gene, PAP I, has been characterized in a few β-, γ- and δ- Proteobacteria . Using the PAP I signature sequence, putative PAPs were identified in bacterial species from the α- and ε- Proteobacteria and from four other bacterial phyla ( Firmicutes , Actinobacteria , Bacteroidetes and Aquificae ). Phylogenetic analysis, alien index and G+C content calculations strongly suggest that the PAPs in the species identified in this study arose by horizontal gene transfer from the β- and γ- Proteobacteria .

Phylogeny and Evolution of RNA 3'-Nucleotidyltransferases in Bacteria.

The tRNA nucleotidyltransferases and poly(A) polymerases belong to a superfamily of nucleotidyltransferases. The amino acid sequences of a number of bacterial tRNA nucleotidyltransferases and poly(A) polymerases have been used to construct a rooted, neighbor-joining phylogenetic tree. Using information gleaned from that analysis, along with data from the rRNA-based phylogenetic tree, structural data available on a number of members of the superfamily and other biochemical information on the superfamily, it is possible to suggest a scheme for the evolution of the bacterial tRNA nucleotidyltransferases and poly(A) polymerases from ancestral species. Elements of that scheme are discussed along with questions arising from the scheme which can be explored experimentally.

Structural and immunological characterization of a new nucleotidyltransferase-like antigen from Paracoccidioides brasiliensis

Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coli BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an αβαβαβ topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (1H-15N HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies.

Antifungal drug ciclopirox olamine reduces HSV-1 replication and disease in mice

Herpes simplex virus (HSV)-1 and HSV-2 cause painful blisters and shallow ulcers in exposed skin and mucosae during primary or recurrent infection. In addition, recurrent and potentially blinding HSV-1 infections of the eye afflict nearly half a million persons in the U.S. Current clinical therapies rely on nucleoside analog drugs such as acyclovir (ACV) or ganciclovir to ameliorate primary infections and reduce the frequency and duration of reactivations. However, these treatments do not fully suppress viral shedding and drug-resistant mutants develop in the eye and in vulnerable, immunosuppressed patients. Herpesvirus DNA replication requires several enzymes in the nucleotidyl transferase superfamily (NTS) that have recombinase and nuclease activities. We previously found that compounds which block NTS enzymes efficiently inhibit replication of HSV-1 and HSV-2 by up to 1 million-fold in Vero and human foreskin fibroblasts. Among the compounds with potent suppressive effects in culture is the anti-fungal drug ciclopirox. Here we report that topical application of ciclopirox olamine to the eyes of mice infected with HSV-1 reduced virus shed from the corneal epithelium compared with saline control, and reduced development of blepharitis to the level of mice treated with ACV. Results were dose-dependent. In addition, treatment with ciclopirox olamine significantly reduced acute and latent HSV-1 infection of the peripheral nervous system. These results support further development of ciclopirox olamine as a repurposed topical agent for HSV infections.

Broad anti-herpesviral activity of α-hydroxytropolones

Herpesviruses are ubiquitous in animals and cause economic losses concomitant with many diseases. Most of the domestic animal herpesviruses are within the subfamily Alphaherpesvirinae, which includes human herpes simplex virus 1 (HSV-1). Suppression of HSV-1 replication has been reported with α-hydroxytropolones (αHTs), aromatic ring compounds that have broad bioactivity due to potent chelating activity. It is postulated that αHTs inhibit enzymes within the nucleotidyltransferase superfamily (NTS). These enzymes require divalent cations for nucleic acid cleavage activity. Potential targets include the nuclease component of the herpesvirus terminase (pUL15C), a highly conserved NTS-like enzyme that cleaves viral DNA into genomic lengths prior to packaging into capsids. Inhibition of pUL15C activity in biochemical assays by various αHTs previously revealed a spectrum of potencies. Interestingly, the most potent anti-pUL15C αHT inhibited HSV-1 replication to a limited extent in cell culture. The aim of this study was to evaluate three different αHT molecules with varying biochemical anti-pUL15C activity for a capacity to inhibit replication of veterinary herpesviruses (BoHV-1, EHV-1, and FHV-1) and HSV-1. Given the known discordant potencies between anti-pUL15C and HSV-1 replication inhibition, a second objective was to elucidate the mechanism of action of these compounds. The results show that αHTs broadly inhibit herpesviruses, with similar inhibitory effect against HSV-1, BoHV-1, EHV-1, and FHV-1. Based on immunoblotting, Southern blotting, and real-time qPCR, the compounds were found to specifically inhibit viral DNA replication. Thus, αHTs represent a new class of broadly active anti-herpesviral compounds with potential veterinary applications.

Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors

The natural product α-hydroxytropolones manicol and β-thujaplicinol inhibit replication of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively) at nontoxic concentrations. Because these were originally developed as divalent metal-sequestering inhibitors of the ribonuclease H activity of HIV-1 reverse transcriptase, α-hydroxytropolones likely target related HSV proteins of the nucleotidyltransferase (NTase) superfamily, which share an "RNase H-like" fold. One potential candidate is pUL15, a component of the viral terminase molecular motor complex, whose C-terminal nuclease domain, pUL15C, has recently been crystallized. Crystallography also provided a working model for DNA occupancy of the nuclease active site, suggesting potential protein-nucleic acid contacts over a region of ∼ 14 bp. In this work, we extend crystallographic analysis by examining pUL15C-mediated hydrolysis of short, closely related DNA duplexes. In addition to defining a minimal substrate length, this strategy facilitated construction of a dual-probe fluorescence assay for rapid kinetic analysis of wild-type and mutant nucleases. On the basis of its proposed role in binding the phosphate backbone, studies with pUL15C variant Lys700Ala showed that this mutation affected neither binding of duplex DNA nor binding of small molecule to the active site but caused a 17-fold reduction in the turnover rate (kcat), possibly by slowing conversion of the enzyme-substrate complex to the enzyme-product complex and/or inhibiting dissociation from the hydrolysis product. Finally, with a view of pUL15-associated nuclease activity as an antiviral target, the dual-probe fluorescence assay, in combination with differential scanning fluorimetry, was used to demonstrate inhibition by several classes of small molecules that target divalent metal at the active site.

Atomistic details of the molecular recognition of DNA-RNA hybrid duplex by ribonuclease H enzyme

Bacillus halodurans (Bh) ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and is a prototypical member of a large family of enzymes that use two-metal ion (Mg2+ or Mn2+) catalysis to cleave nucleic acids. Long timescale molecular dynamics simulations have been performed on the BhRNase H-DNA-RNA hybrid complex and the respective monomers to understand the recognition mechanism, conformational preorganization, active site dynamics and energetics involved in the complex formation. Several structural and energetic analyses were performed and significant structural changes are observed in enzyme and hybrid duplex during complex formation. Hybrid molecule binding to RNase H enzyme leads to conformational changes in the DNA strand. The ability of the DNA strand in the hybrid duplex to sample conformations corresponding to typical A- and B-type nucleic acids and the characteristic minor groove widthseem to be crucial for efficient binding. Sugar moieties in certain positions interacting with the protein structure undergo notable conformational transitions. The water coordination and arrangement around the metal ions in active site region are quite stable, suggesting their important role in enzymatic catalysis. Details of key interactions found at the interface of enzyme-nucleic acid complex that are responsible for its stability are discussed.

Inhibitors of nucleotidyltransferase superfamily enzymes suppress herpes simplex virus replication.

Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 μM, with suppression at 50 μM reaching ∼1 million-fold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 μM with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family.

Crystal structure confirmation of JHP933 as a nucleotidyltransferase superfamily protein from Helicobacter pylori strain J99.

Helicobacter pylori is a well-known pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Recently, there has been more considerable interest in strain-specific genes located in plasticity regions with great genetic variability. However, little is known about many of these genes. Studies suggested that certain genes in this region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. JHP933, a conserved putative protein of unknown function, is encoded by the gene in plasticity region of H. pylori strain J99. Here we have determined the structure of JHP933. Our work demonstrates that JHP933 is a nucleotidyltransferase superfamily protein with a characteristic αβαβαβα topology. A superposition demonstrates overall structural homology of the JHP933 N-terminal fragment with lincosamide antibiotic adenylyltransferase LinA and identifies a possible substrate-binding cleft of JHP933. Furthermore, through structural comparison with LinA and LinB, we pinpoint conservative active site residues which may contribute to divalent ion coordination and substrate binding.

Kinetic Analysis of DNA Strand Joining by Chlorella Virus DNA Ligase and the Role of Nucleotidyltransferase Motif VI in Ligase Adenylylation

Chlorella virus DNA ligase (ChVLig) is an instructive model for mechanistic studies of the ATP-dependent DNA ligase family. ChVLig seals 3'-OH and 5'-PO(4) termini via three chemical steps: 1) ligase attacks the ATP α phosphorus to release PP(i) and form a covalent ligase-adenylate intermediate; 2) AMP is transferred to the nick 5'-phosphate to form DNA-adenylate; 3) the 3'-OH of the nick attacks DNA-adenylate to join the polynucleotides and release AMP. Each chemical step requires Mg(2+). Kinetic analysis of nick sealing by ChVLig-AMP revealed that the rate constant for phosphodiester synthesis (k(step3) = 25 s(-1)) exceeds that for DNA adenylylation (k(step2) = 2.4 s(-1)) and that Mg(2+) binds with similar affinity during step 2 (K(d) = 0.77 mM) and step 3 (K(d) = 0.87 mM). The rates of DNA adenylylation and phosphodiester synthesis respond differently to pH, such that step 3 becomes rate-limiting at pH ≤ 6.5. The pH profiles suggest involvement of one and two protonation-sensitive functional groups in catalysis of steps 2 and 3, respectively. We suggest that the 5'-phosphate of the nick is the relevant protonation-sensitive moiety and that a dianionic 5'-phosphate is necessary for productive step 2 catalysis. Motif VI, located at the C terminus of the OB-fold domain of ChVLig, is a conserved feature of ATP-dependent DNA ligases and GTP-dependent mRNA capping enzymes. Presteady state and burst kinetic analysis of the effects of deletion and missense mutations highlight the catalytic contributions of ChVLig motif VI, especially the Asp-297 carboxylate, exclusively during the ligase adenylylation step.

Virtual High-Throughput Screening Identifies Mycophenolic Acid as a Novel RNA Capping Inhibitor

The RNA guanylyltransferase (GTase) is involved in the synthesis of the m7Gppp-RNA cap structure found at the 5′ end of eukaryotic mRNAs. GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases. GTases catalyze a two-step reaction in which they initially utilize GTP as a substrate to form a covalent enzyme-GMP intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the Gppp-RNA structure. In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. Using this approach, we demonstrate that mycophenolic acid (MPA) can inhibit the GTase reaction by preventing the catalytic transfer of the GMP moiety onto an acceptor RNA. As such, MPA represents a novel type of inhibitor against RNA guanylyltransferases that inhibits the second step of the catalytic reaction. Moreover, we show that the addition of MPA to S. cerevisiae cells leads to a reduction of capped mRNAs. Finally, biochemical assays also demonstrate that MPA can inhibit DNA ligases through inhibition of the second step of the reaction. The biological implications of these findings for the MPA-mediated inhibition of members of the covalent nucleotidyl superfamily are discussed.

Functional Dissection of the DNA Interface of the Nucleotidyltransferase Domain of Chlorella Virus DNA Ligase

Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3'-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3'-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3'-OH nucleoside in the catalysis of DNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig.

Mechanism for the Alteration of the Substrate Specificities of Template-Independent RNA Polymerases

PolyA polymerase (PAP) adds a polyA tail onto the 3'-end of RNAs without a nucleic acid template, using adenosine-5'-triphosphate (ATP) as a substrate. The mechanism for the substrate selection by eubacterial PAP remains obscure. Structural and biochemical studies of Escherichia coli PAP (EcPAP) revealed that the shape and size of the nucleobase-interacting pocket of EcPAP are maintained by an intra-molecular hydrogen-network, making it suitable for the accommodation of only ATP, using a single amino acid, Arg(197). The pocket structure is sustained by interactions between the catalytic domain and the RNA-binding domain. EcPAP has a flexible basic C-terminal region that contributes to optimal RNA translocation for processive adenosine 5'-monophosphate (AMP) incorporations onto the 3'-end of RNAs. A comparison of the EcPAP structure with those of other template-independent RNA polymerases suggests that structural changes of domain(s) outside the conserved catalytic core domain altered the substrate specificities of the template-independent RNA polymerases.

Understanding the Effect of Magnesium Ion Concentration on the Catalytic Activity of Ribonuclease H through Computation: Does a Third Metal Binding Site Modulate Endonuclease Catalysis?

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg(2+) concentration. Moreover, in RNase H, the glutamate residue E188 has been shown to be essential for full enzymatic activation, regardless of the Mg(2+) concentration. The catalytic center is known to contain two Mg(2+) ions, and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg(2+). Importantly, the presence of a third Mg(2+) ion, bound to the second-shell ligand E188, is a persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations, depending on the concentration of Mg(2+). At standard concentration, a third Mg(2+) is found in the catalytic pocket, but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg(2+) ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg(2+) ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A and also supports the key role of E188 in localizing the third Mg(2+) ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calculations support the hypothesis that RNase H can accommodate three divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.

Structure and function of the polymerase core of TRAMP, a RNA surveillance complex.

The Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex recognizes aberrant RNAs in Saccharomyces cerevisiae and targets them for degradation. A TRAMP subcomplex consisting of a noncanonical poly(A) RNA polymerase in the Pol ss superfamily of nucleotidyl transferases, Trf4p, and a zinc knuckle protein, Air2p, mediates initial substrate recognition. Trf4p and related eukaryotic poly(A) and poly(U) polymerases differ from other characterized enzymes in the Pol ss superfamily both in sequence and in the lack of recognizable nucleic acid binding motifs. Here we report, at 2.7-A resolution, the structure of Trf4p in complex with a fragment of Air2p comprising two zinc knuckle motifs. Trf4p consists of a catalytic and central domain similar in fold to those of other noncanonical Pol beta RNA polymerases, and the two zinc knuckle motifs of Air2p interact with the Trf4p central domain. The interaction surface on Trf4p is highly conserved across eukaryotes, providing evidence that the Trf4p/Air2p complex is conserved in higher eukaryotes as well as in yeast and that the TRAMP complex may also function in RNA surveillance in higher eukaryotes. We show that Air2p, and in particular sequences encompassing a zinc knuckle motif near its N terminus, modulate Trf4p activity, and we present data supporting a role for this zinc knuckle in RNA binding. Finally, we show that the RNA 3' end plays a role in substrate recognition.

Atomistic details of the associative phosphodiester cleavage in human ribonuclease H

During translation of the genetic information of DNA into proteins, mRNA is synthesized by RNA polymerase and after the transcription process degraded by RNase H. The endoribonuclease RNase H is a member of the nucleotidyl-transferase (NT) superfamily and is known to hydrolyze the phosphodiester bonds of RNA which is hybridized to DNA. Retroviral RNase H is part of the viral reverse transcriptase enzyme that is indispensable for the proliferation of retroviruses, such as HIV. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS. In our study we investigated the molecular mechanism of RNA cleavage by human RNase H using a comprehensive high level DFT/B3LYP QM/MM theoretical method for the calculation of the stationary points and nudged elastic band (NEB) and free energy calculations to identify the transition state structures, the rate limiting step and the reaction barrier. Our calculations reveal that the catalytic mechanism proceeds in two steps and that the nature of the nucleophile is a water molecule. In the first step, the water attack on the scissile phosphorous is followed by a proton transfer from the water to the O2P oxygen and a trigonal bipyramidal pentacoordinated phosphorane is formed. Subsequently, in the second step the proton is shuttled to the O3' oxygen to generate the product state. During the reaction mechanism two Mg(2+) ions support the formation of a stable associated in-line S(N)2-type phosphorane intermediate. Our calculated energy barrier of 19.3 kcal mol(-1) is in excellent agreement with experimental findings (20.5 kcal mol(-1)). These results may contribute to the clarification and understanding of the RNase H reaction mechanism and of further enzymes from the RNase family.

CUCU Modification of mRNA Promotes Decapping and Transcript Degradation in Aspergillus nidulans

In eukaryotes, mRNA decay is generally initiated by the shortening of the poly(A) tail mediated by the major deadenylase complex Ccr4-Caf1-Not. The deadenylated transcript is then rapidly degraded, primarily via the decapping-dependent pathway. Here we report that in Aspergillus nidulans both the Caf1 and Ccr4 orthologues are functionally distinct deadenylases in vivo: Caf1 is required for the regulated degradation of specific transcripts, and Ccr4 is responsible for basal degradation. Intriguingly disruption of the Ccr4-Caf1-Not complex leads to deadenylation-independent decapping. Additionally, decapping is correlated with a novel transcript modification, addition of a CUCU sequence. A member of the nucleotidyltransferase superfamily, CutA, is required for this modification, and its disruption leads to a reduced rate of decapping and subsequent transcript degradation. We propose that 3' modification of adenylated mRNA, which is likely to represent a common eukaryotic process, primes the transcript for decapping and efficient degradation.

Structure and Mechanism of the Lincosamide Antibiotic Adenylyltransferase LinB

Lincosamides make up an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. Predictably, lincosamide-resistant microorganisms have emerged with antibiotic modification as one of their major resistance strategies. Inactivating enzymes LinB/A catalyze adenylylation of the drug; however, little is known about their mechanistic and structural properties. We determined two X-ray structures of LinB: ternary substrate- and binary product-bound complexes. Structural and kinetic characterization of LinB, mutagenesis, solvent isotope effect, and product inhibition studies are consistent with a mechanism involving direct in-line nucleotidyl transfer. The characterization of LinB enabled its classification as a member of a nucleotidyltransferase superfamily, along with nucleotide polymerases and aminoglycoside nucleotidyltransferases, and this relationship offers further support for the LinB mechanism. The LinB structure provides an evolutionary link to ancient nucleotide polymerases and suggests that, like protein kinases and acetyltransferases, these are proto-resistance elements from which drug resistance can evolve.

Discovery and design of DNA and RNA ligase inhibitors in infectious microorganisms

Background: Members of the nucleotidyltransferase superfamily known as DNA and RNA ligases carry out the enzymatic process of polynucleotide ligation. These guardians of genomic integrity share a three-step ligation mechanism, as well as common core structural elements. Both DNA and RNA ligases have experienced a surge of recent interest as chemotherapeutic targets for the treatment of a range of diseases, including bacterial infection, cancer and the diseases caused by the protozoan parasites known as trypanosomes. Objective: In this review, we will focus on efforts targeting pathogenic microorganisms; specifically, bacterial NAD+-dependent DNA ligases, which are promising broad-spectrum antibiotic targets, and ATP-dependent RNA-editing ligases from Trypanosoma brucei, the species responsible for the devastating neurodegenerative disease, African sleeping sickness. Conclusion: High-quality crystal structures of both NAD+-dependent DNA ligase and the T. brucei RNA-editing ligase have facilitated the development of a number of promising leads. For both targets, further progress will require surmounting permeability issues and improving selectivity and affinity.

Toward Understanding the Conformational Dynamics of RNA Ligation

Members of the genus Trypanosoma, which include the pathogenic species Trypanosoma brucei and Trypanosoma cruzi, edit their post-transcriptional mitochondrial RNA via a multiprotein complex called the editosome. In T. brucei, the RNA is nicked prior to uridylate insertion and deletion. Following editing, nicked RNA is religated by one of two RNA-editing ligases (TbREL). This study describes a recent 70 ns molecular dynamics simulation of TbREL1, an ATP-dependent RNA-editing ligase of the nucleotidyltransferase superfamily that is required for the survival of T. brucei insect and bloodstream forms. In this work, a model of TbREL1 in complex with its full double-stranded RNA (dsRNA) substrate is created on the basis of the homologous relation between TbREL1 and T4 Rnl2. The simulation captures TbREL1 dynamics in the state immediately preceding RNA ligation, providing insights into the functional dynamics and catalytic mechanism of the kinetoplastid ligation reaction. Important features of RNA binding and specificity are revealed for kinetoplastid ligases and the broader nucleotidyltransferase superfamily.