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Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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structures of atp bound dna Ligase d in a closed domain conformation reveal a network of amino acid and metal contacts to the atp phosphates
Journal of Biological Chemistry, 2019Co-Authors: Mihaelacarmen Unciuleac, Yehuda Goldgur, Stewart ShumanAbstract:DNA Ligases are the sine qua non of genome integrity and essential for DNA replication and repair in all organisms. DNA Ligases join 3'-OH and 5'-PO4 ends via a series of three nucleotidyl transfer steps. In step 1, Ligase reacts with ATP or NAD+ to form a covalent Ligase-(lysyl-Nζ)-AMP intermediate and release pyrophosphate (PPi) or nicotinamide mononucleotide. In step 2, AMP is transferred from Ligase-adenylate to the 5'-PO4 DNA end to form a DNA-adenylate intermediate (AppDNA). In step 3, Ligase catalyzes attack by a DNA 3'-OH on the DNA-adenylate to seal the two ends via a phosphodiester bond and release AMP. Eukaryal, archaeal, and many bacterial and viral DNA Ligases are ATP-dependent. The catalytic core of ATP-dependent DNA Ligases consists of an N-terminal nucleotidyltransferase domain fused to a C-terminal OB domain. Here we report crystal structures at 1.4-1.8 A resolution of Mycobacterium tuberculosis LigD, an ATP-dependent DNA Ligase dedicated to nonhomologous end joining, in complexes with ATP that highlight large movements of the OB domain (∼50 A), from a closed conformation in the ATP complex to an open conformation in the covalent Ligase-AMP intermediate. The LigD·ATP structures revealed a network of amino acid contacts to the ATP phosphates that stabilize the transition state and orient the PPi leaving group. A complex with ATP and magnesium suggested a two-metal mechanism of lysine adenylylation driven by a catalytic Mg2+ that engages the ATP α phosphate and a second metal that bridges the ATP β and γ phosphates.
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crucial role for dna Ligase iii in mitochondria but not in xrcc1 dependent repair
Nature, 2011Co-Authors: Stewart Shuman, Deniz Simsek, Amy M Furda, Jerome Artus, Erika Brunet, Annakaterina Hadjantonakis, Bennett Van HoutenAbstract:Mammalian cells contain three different DNA Ligase enzymes, each with different properties but all involved in DNA replication and repair. Ligase III (Lig3) is known to form a complex with the nuclear DNA repair protein Xrcc1, and Lig3 null animals cannot be made. This raises the question of whether this nuclear role in base-excision repair (BER) is the critical function of Lig3 that maintains viability. Two groups reporting in this issue of Nature investigate different aspects of Lig3 function in vivo, both concluding that the catalytic activity of Lig3 is critical for mitochondrial DNA maintenance and viability, but unexpectedly, is dispensable for Xrcc1-mediated nuclear BER. These findings suggest that Lig3 mutations might cause some of the human syndromes associated with defects in the replication and/or repair of mitochondrial DNA. Eukaryotic cells have several DNA Ligases. DNA Ligase III (Lig3) forms a complex with Xrcc1 that can function in nuclear repair. But, Lig3 null animals cannot be made; is this nuclear role in base excision repair its critical function? This is one of two papers showing that the role of Lig3 in the nucleus is non-essential. Rather, the catalytic activity of Lig3, but not Xrcc1, is essential for the maintenance of mitochondria. Mammalian cells have three ATP-dependent DNA Ligases, which are required for DNA replication and repair1. Homologues of Ligase I (Lig1) and Ligase IV (Lig4) are ubiquitous in Eukarya, whereas Ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein Xrcc1 (ref. 1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality2, which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (mES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCA1 carboxy (C)-terminal-related (BRCT) domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA Ligase, the minimal eukaryal nick-sealing enzyme3, or Escherichia coli LigA, an NAD+-dependent Ligase1. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells4,5,6. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.
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DNA Ligases: Progress and Prospects
Journal of Biological Chemistry, 2009Co-Authors: Stewart ShumanAbstract:DNA Ligases seal 5′-PO4 and 3′-OH polynucleotide ends via three nucleotidyl transfer steps involving Ligase-adenylate and DNA-adenylate intermediates. DNA Ligases are essential guardians of genomic integrity, and Ligase dysfunction underlies human genetic disease syndromes. Crystal structures of DNA Ligases bound to nucleotide and nucleic acid substrates have illuminated how Ligase reaction chemistry is catalyzed, how Ligases recognize damaged DNA ends, and how protein domain movements and active-site remodeling are used to choreograph the end-joining pathway. Although a shared feature of DNA Ligases is their envelopment of the nicked duplex as a C-shaped protein clamp, they accomplish this feat by using remarkably different accessory structural modules and domain topologies. As structural, biochemical, and phylogenetic insights coalesce, we can expect advances on several fronts, including (i) pharmacological targeting of Ligases for antibacterial and anticancer therapies and (ii) the discovery and design of new strand-sealing enzymes with unique substrate specificities.
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Structure and Mechanism of RNA Ligase
Structure, 2004Co-Authors: Li Kai Wang, Christopher D. Lima, Stewart ShumanAbstract:T4 RNA Ligase 2 (Rnl2) exemplifies an RNA Ligase family that includes the RNA editing Ligases (RELs) of Trypanosoma and Leishmania . The Rnl2/REL enzymes are defined by essential signature residues and a unique C-terminal domain, which we show is essential for sealing of 3′-OH and 5′-PO 4 RNA ends by Rnl2, but not for Ligase adenylation or phosphodiester bond formation at a preadenylated AppRNA end. The N-terminal segment Rnl2(1-249) of the 334 aa Rnl2 protein comprises an autonomous adenylyltransferase/AppRNA Ligase domain. We report the 1.9 A crystal structure of the Ligase domain with AMP bound at the active site, which reveals a shared fold, catalytic mechanism, and evolutionary history for RNA Ligases, DNA Ligases, and mRNA capping enzymes.
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Structure-function analysis of T4 RNA Ligase 2.
Journal of Biological Chemistry, 2003Co-Authors: Shenmin Yin, Stewart ShumanAbstract:Abstract Bacteriophage T4 RNA Ligase 2 (Rnl2) exemplifies a polynucleotide Ligase family that includes the trypanosome RNA-editing Ligases and putative RNA Ligases encoded by eukaryotic viruses and archaea. Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining. We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of Ligase adenylylation and phosphodiester bond formation. The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates. Our mutagenesis results underscore a shared active site architecture in Rnl2-like Ligases, DNA Ligases, and mRNA capping enzymes. They also highlight two essential signature residues, Glu34 and Asn40, that flank the active site lysine nucleophile (Lys35) and are unique to the Rnl2-like Ligase family.
Ravishankar Ramachandran - One of the best experts on this subject based on the ideXlab platform.
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Tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can specifically target bacterial DNA Ligases and can distinguish them from human DNA Ligase I.
Organic and Biomolecular Chemistry, 2015Co-Authors: Nisha Yadav, Taran Khanam, Ankita Shukla, Kanchan Hajela, Ravishankar RamachandranAbstract:DNA Ligases are critical components for DNA metabolism in all organisms. NAD+-dependent DNA Ligases (LigA) found exclusively in bacteria and certain entomopoxviruses are drawing increasing attention as therapeutic targets as they differ in their cofactor requirement from ATP-dependent eukaryotic homologs. Due to the similarities in the cofactor binding sites of the two classes of DNA Ligases, it is necessary to find determinants that can distinguish between them for the exploitation of LigA as an anti-bacterial target. In the present endeavour, we have synthesized and evaluated a series of tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives for their ability to distinguish between bacterial and human DNA Ligases. The in vivo inhibition assays that employed LigA deficient E. coli GR501 and S. typhimurium LT2 bacterial strains, rescued by ATP-dependent T4 DNA Ligase or Mycobacterium tuberculosis NAD+-dependent DNA Ligase (Mtb LigA), respectively, showed that the compounds can specifically inhibit bacterial LigA. The in vitro enzyme inhibition assays using purified MtbLigA, human DNA Ligase I & T4 DNA Ligase showed specific inhibition of MtbLigA at low micromolar range. Our results demonstrate that tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can distinguish between bacterial and human DNA Ligases by ∼5-folds. In silico docking and enzyme inhibition assays identified that the compounds bind to the cofactor binding site and compete with the cofactor. Ethidium bromide displacement and gel-shift assays showed that the inhibitors do not exhibit any unwanted general interactions with the substrate DNA. These results set the stage for the detailed exploration of this compound class for development as antibacterials.
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Tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can specifically target bacterial DNA Ligases and can distinguish them from human DNA Ligase I.
Organic & Biomolecular Chemistry, 2015Co-Authors: Nisha Yadav, Taran Khanam, Ankita Shukla, Kanchan Hajela, Niyati Rai, Ravishankar RamachandranAbstract:DNA Ligases are critical components for DNA metabolism in all organisms. NAD+-dependent DNA Ligases (LigA) found exclusively in bacteria and certain entomopoxviruses are drawing increasing attention as therapeutic targets as they differ in their cofactor requirement from ATP-dependent eukaryotic homologs. Due to the similarities in the cofactor binding sites of the two classes of DNA Ligases, it is necessary to find determinants that can distinguish between them for the exploitation of LigA as an anti-bacterial target. In the present endeavour, we have synthesized and evaluated a series of tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives for their ability to distinguish between bacterial and human DNA Ligases. The in vivo inhibition assays that employed LigA deficient E. coli GR501 and S. typhimurium LT2 bacterial strains, rescued by ATP-dependent T4 DNA Ligase or Mycobacterium tuberculosis NAD+-dependent DNA Ligase (Mtb LigA), respectively, showed that the compounds can specifically inhibit bacterial LigA. The in vitro enzyme inhibition assays using purified MtbLigA, human DNA Ligase I & T4 DNA Ligase showed specific inhibition of MtbLigA at low micromolar range. Our results demonstrate that tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can distinguish between bacterial and human DNA Ligases by ∼5-folds. In silico docking and enzyme inhibition assays identified that the compounds bind to the cofactor binding site and compete with the cofactor. Ethidium bromide displacement and gel-shift assays showed that the inhibitors do not exhibit any unwanted general interactions with the substrate DNA. These results set the stage for the detailed exploration of this compound class for development as antibacterials.
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nad dependent dna Ligase rv3014c from mycobacterium tuberculosis crystal structure of the adenylation domain and identification of novel inhibitors
Journal of Biological Chemistry, 2005Co-Authors: Sandeep Kumar Srivastava, Rama P Tripathi, Ravishankar RamachandranAbstract:Abstract DNA Ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for Ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent Ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent Ligases as evidenced by in vitro assays using T4 Ligase and human DNA Ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive Ligase mutant and a Ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential Ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.
Alan E. Tomkinson - One of the best experts on this subject based on the ideXlab platform.
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Altered DNA Ligase activity in human disease.
Mutagenesis, 2019Co-Authors: Alan E. Tomkinson, Tasmin Naila, Seema Khattri BhandariAbstract:The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA Ligases. DNA Ligases I, III and IV each function in the nucleus whereas DNA Ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA Ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA Ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA Ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA Ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA Ligase I deficiency is not known. Overexpression of each of the DNA Ligases has been observed in cancers. For DNA Ligase I, this reflects increased proliferation. Elevated levels of DNA Ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA Ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA Ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.
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scr7 is neither a selective nor a potent inhibitor of human dna Ligase iv
DNA Repair, 2016Co-Authors: George E Greco, Yoshihiro Matsumoto, Rhys Brooks, Zhengfei Lu, Michael R Lieber, Alan E. TomkinsonAbstract:DNA Ligases are attractive therapeutics because of their involvement in completing the repair of almost all types of DNA damage. A series of DNA Ligase inhibitors with differing selectivity for the three human DNA Ligases were identified using a structure-based approach with one of these inhibitors being used to inhibit abnormal DNA Ligase IIIα-dependent repair of DNA double-strand breaks (DSB)s in breast cancer, neuroblastoma and leukemia cell lines. Raghavan and colleagues reported the characterization of a derivative of one of the previously identified DNA Ligase inhibitors, which they called SCR7 (designated SCR7-R in our experiments using SCR7). SCR7 appeared to show increased selectivity for DNA Ligase IV, inhibit the repair of DSBs by the DNA Ligase IV-dependent non-homologous end-joining (NHEJ) pathway, reduce tumor growth, and increase the efficacy of DSB-inducing therapeutic modalities in mouse xenografts. In attempting to synthesize SCR7, we encountered problems with the synthesis procedures and discovered discrepancies in its reported structure. We determined the structure of a sample of SCR7 and a related compound, SCR7-G, that is the major product generated by the published synthesis procedure for SCR7. We also found that SCR7-G has the same structure as the compound (SCR7-X) available from a commercial vendor (XcessBio). The various SCR7 preparations had similar activity in DNA ligation assay assays, exhibiting greater activity against DNA Ligases I and III than DNA Ligase IV. Furthermore, SCR7-R failed to inhibit DNA Ligase IV-dependent V(D)J recombination in a cell-based assay. Based on our results, we conclude that SCR7 and the SCR7 derivatives are neither selective nor potent inhibitors of DNA Ligase IV.
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Structure and function of the DNA Ligases encoded by the mammalian LIG3 gene.
Gene, 2013Co-Authors: Alan E. Tomkinson, Annahita SallmyrAbstract:Among the mammalian genes encoding DNA Ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA Ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA Ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA Ligase IIIα and DNA Ligase I in excision repair. In addition, DNA Ligase IIIα is essential for DNA replication in the absence of the replicative DNA Ligase, DNA Ligase I. DNA Ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA Ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA Ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA Ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.
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Eukaryotic DNA Ligases: structural and functional insights.
Annual Review of Biochemistry, 2008Co-Authors: Alan E. TomkinsonAbstract:DNA Ligases are required for DNA replication, repair, and recombination. In eukaryotes, there are three families of ATP-dependent DNA Ligases. Members of the DNA Ligase I and IV families are found in all eukaryotes, whereas DNA Ligase III family members are restricted to vertebrates. These enzymes share a common catalytic region comprising a DNA-binding domain, a nucleotidyltransferase (NTase) domain, and an oligonucleotide/oligosaccharide binding (OB)-fold domain. The catalytic region encircles nicked DNA with each of the domains contacting the DNA duplex. The unique segments adjacent to the catalytic region of eukaryotic DNA Ligases are involved in specific protein-protein interactions with a growing number of DNA replication and repair proteins. These interactions determine the specific cellular functions of the DNA Ligase isozymes. In mammals, defects in DNA ligation have been linked with an increased incidence of cancer and neurodegeneration.
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rational design of human dna Ligase inhibitors that target cellular dna replication and repair
Cancer Research, 2008Co-Authors: Xi Chen, Shijun Zhong, Barbara Dziegielewska, Gerald M Wilson, Alexander D Mackerell, Alan E. TomkinsonAbstract:Based on the crystal structure of human DNA Ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA Ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA Ligase I. Notably, a subset of these compounds was also active against the other human DNA Ligases. Three compounds that differed in their specificity for the three human DNA Ligases were analyzed further. L82 inhibited DNA Ligase I, L67 inhibited DNA Ligases I and III, and L189 inhibited DNA Ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA Ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the Ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA Ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents. [Cancer Res 2008;68(9):3169–77]
Judit Ovadi - One of the best experts on this subject based on the ideXlab platform.
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halotag targeted sirtuin rearranging ligand sirreal for the development of proteolysis targeting chimeras protacs against the lysine deacetylase sirtuin 2 sirt2
ChemBioChem, 2020Co-Authors: Matthias Schiedel, Nathalie Wossner, Attila Lehotzky, Sandor Szunyogh, Soren Hammelmann, Dina Robaa, Wolfgang Sippl, Judit Oláh, Oliver Einsle, Judit OvadiAbstract:We have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin Ligase cereblon to form a so-called proteolysis targeting chimera (PROTAC), enabled small molecule-induced degradation of Sirt2. Here, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin Ligases. Using this orthogonal approach for Sirt2 degradation, we show that also other E3 Ligases than cereblon, such as the E3 ubiquitin Ligase parkin, can be harnessed for small molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be utilized as an E3 Ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.
Matthias Schiedel - One of the best experts on this subject based on the ideXlab platform.
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halotag targeted sirtuin rearranging ligand sirreal for the development of proteolysis targeting chimeras protacs against the lysine deacetylase sirtuin 2 sirt2
ChemBioChem, 2020Co-Authors: Matthias Schiedel, Nathalie Wossner, Attila Lehotzky, Sandor Szunyogh, Soren Hammelmann, Dina Robaa, Wolfgang Sippl, Judit Oláh, Oliver Einsle, Judit OvadiAbstract:We have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin Ligase cereblon to form a so-called proteolysis targeting chimera (PROTAC), enabled small molecule-induced degradation of Sirt2. Here, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin Ligases. Using this orthogonal approach for Sirt2 degradation, we show that also other E3 Ligases than cereblon, such as the E3 ubiquitin Ligase parkin, can be harnessed for small molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be utilized as an E3 Ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.