Dumbbell DNA-templated CuNPs as a nano-fluorescent probe for detection of enzymes involved in ligase-mediated DNA repair.

DNA repair processes are responsible for maintaining genome stability. Ligase and polynucleotide kinase (PNK) have important roles in ligase-mediated DNA repair. The development of analytical methods to monitor these enzymes involved in DNA repair pathways is of great interest in biochemistry and biotechnology. In this work, we reported a new strategy for label-free monitoring PNK and ligase activity by using dumbbell-shaped DNA templated copper nanoparticles (CuNPs). In the presence of PNK and ligase, the dumbbell-shaped DNA probe (DP) was locked and could resist the digestion of exonucleases and then served as an efficient template for synthesizing fluorescent CuNPs. However, in the absence of ligase or PNK, the nicked DP could be digested by exonucleases and failed to template fluorescent CuNPs. Therefore, the fluorescence changes of CuNPs could be used to evaluate these enzymes activity. Under the optimal conditions, highly sensitive detection of ligase activity of about 1U/mL and PNK activity down to 0.05U/mL is achieved. To challenge the practical application capability of this strategy, the detection of analyte in dilute cells extracts was also investigated and showed similar linear relationships. In addition to ligase and PNK, this sensing strategy was also extended to the detection of phosphatase, which illustrates the versatility of this strategy.

A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.

DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.

Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology.

With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area.

Base-modified NAD and AMP derivatives and their activity against bacterial DNA ligases.

We report the chemical synthesis and conformational analysis of a collection of 2-, 6- and 8-substituted derivatives of ß-NAD(+) and AMP, and their biochemical evaluation against NAD(+)-dependent DNA ligases from Escherichia coli and Mycobacterium tuberculosis. Bacterial DNA ligases are validated anti-microbial targets, and new strategies for their inhibition are therefore of considerable scientific and practical interest. Our study includes several pairs of ß-NAD(+) and AMP derivatives with the same substitution pattern at the adenine base. This has enabled the first direct comparison of co-substrate and inhibitor behaviour against bacterial DNA ligases. Our results suggest that an additional substituent in position 6 or 8 of the adenine base in ß-NAD(+) is detrimental for activity as either co-substrate or inhibitor. In contrast, substituents in position 2 are not only tolerated, but appear to give rise to a new mode of inhibition, which targets the conformational changes these DNA ligases undergo during catalysis. Using a molecular modelling approach, we highlight that these findings have important implications for our understanding of ligase mechanism and inhibition, and may provide a promising starting point for the rational design of a new class of inhibitors against NAD(+)-dependent DNA ligases.

Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.

The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.

Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida.

The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATP-dependent DNA ligase in addition to a housekeeping NAD-dependent enzyme. In order to study the structure and activity of the ATP dependent ligase in vitro we have undertaken its recombinant production and purification from an Escherichia coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second it was necessary to remove the large amounts of E. coli DNA present in bacterial lysates without contamination of the protein preparation by nucleases which might interfere with future assaying. The toxicity problem was overcome by fusion of the putative ligase to large solubility tags such as maltose-binding protein (MBP) or Glutathione-S-transferase (GST), and DNA was removed by treatment with a nuclease which could be inhibited by reducing agents. As the A. salmonicida ATP-dependent DNA ligase gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced. Both possessed ATP-dependent DNA ligase activity, but the truncated form was significantly more active. Here we detail the first reported production, purification and preliminary characterization of active A. salmonicida ATP-dependent DNA ligase.

The minimal Bacillus subtilis nonhomologous end joining repair machinery.

It is widely accepted that repair of double-strand breaks in bacteria that either sporulate or that undergo extended periods of stationary phase relies not only on homologous recombination but also on a minimal nonhomologous end joining (NHEJ) system consisting of a dedicated multifunctional ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku. Bacillus subtilis is one of the bacterial members with a NHEJ system that contributes to genome stability during the stationary phase and germination of spores, having been characterized exclusively in vivo. Here, the in vitro analysis of the functional properties of the purified B. subtilis LigD (BsuLigD) and Ku (BsuKu) proteins is presented. The results show that the essential biochemical signatures exhibited by BsuLigD agree with its proposed function in NHEJ: i) inherent polymerization activity showing preferential insertion of NMPs, ii) specific recognition of the phosphate group at the downstream 5' end, iii) intrinsic ligase activity, iv) ability to promote realignments of the template and primer strands during elongation of mispaired 3' ends, and v) it is recruited to DNA by BsuKu that stimulates the inherent polymerization and ligase activities of the enzyme allowing it to deal with and to hold different and unstable DNA realignments.

Structure of the catalytic region of DNA ligase IV in complex with an Artemis fragment sheds light on double-strand break repair.

Nonhomologous end joining (NHEJ) is central to the repair of double-stranded DNA breaks throughout the cell cycle and plays roles in the development of the immune system. Although three-dimensional structures of most components of NHEJ have been defined, those of the catalytic region of DNA ligase IV (LigIV), a specialized DNA ligase known to work in NHEJ, and of Artemis have remained unresolved. Here, we report the crystal structure at 2.4 Å resolution of the catalytic region of LigIV (residues 1-609) in complex with an Artemis peptide. We describe interactions of the DNA-binding domain of LigIV with the continuous epitope of Artemis, which, together, form a three-helix bundle. A kink in the first helix of LigIV introduced by a conserved VPF motif gives rise to a hydrophobic pocket, which accommodates a conserved tryptophan from Artemis. We provide structural insights into features of LigIV among human DNA ligases.

Expression, purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase.

We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg(2+) and Mn(2+). The optimal concentrations of ATP cofactor and Mg(2+) ion were 0.01-2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5' to the nick inhibited ligation more than those at the first position 3' to the nick. The mismatches at other positions 5' to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5' to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5' to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.

Expression, purification, crystallization and preliminary crystallographic analysis of a thermostable DNA ligase from the archaeon Thermococcus sibiricus.

DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential to maintain the integrity of the genome in DNA replication, recombination and repair. A recombinant ATP-dependent DNA ligase from the hyperthermophilic anaerobic archaeon Thermococcus sibiricus was expressed in Escherichia coli and purified. Crystals were grown by vapour diffusion using the hanging-drop method with 17%(w/v) PEG 4000 and 8.5%(v/v) 2-propanol as precipitants. A diffraction experiment was performed with a single crystal, which diffracted X-rays to 3.0 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 58.590, b = 87.540, c = 126.300 Å.

Structure guided understanding of NAD+ recognition in bacterial DNA ligases.

NAD(+)-dependent DNA ligases (LigA) are essential bacterial enzymes that catalyze phosphodiester bond formation during DNA replication and repair processes. Phosphodiester bond formation proceeds through a 3-step reaction mechanism. In the first step, the LigA adenylation domain interacts with NAD(+) to form a covalent enzyme-AMP complex. Although it is well established that the specificity for binding of NAD(+) resides within the adenylation domain, the precise recognition elements for the initial binding event remain unclear. We report here the structure of the adenylation domain from Haemophilus influenzae LigA. This structure is a first snapshot of a LigA-AMP intermediate with NAD(+) bound to domain 1a in its open conformation. The binding affinities of NAD(+) for adenylated and nonadenylated forms of the H. influenzae LigA adenylation domain were similar. The combined crystallographic and NAD(+)-binding data suggest that the initial recognition of NAD(+) is via the NMN binding region in domain 1a of LigA.

[Cloning of genes, purification and properties investigation of recombinant DNA ligases from the thermophilic archaeon Pyrococcus abyssi and Methanobacterium thermoautotrophicum].

The genes encoding of DNA ligases from the thermophilic archaeon Pyrococcus abyssi (PabDNA ligase) and Methanobacterium thermoautotrophicum (MthDNA ligase) were cloned and expressed in Escherichia coli. The activity of purified enzymes was studied by ligation of two oligonucleotides, one of which had preformed hairpin structure. In the used system the maximal output of reaction products for both DNA ligases was observed near 70 degrees C that is explained by substrate thermostability. At stoichiometric ratio of enzymes and substrate the output of a product reaches of plateau at 70-75% of theoretical ones. Investigated DNA ligases showed different thermostability. The half-time life of PabDNA ligase was about 60 min at 90 degrees C. MthDNA ligase was completely inactivated at this temperature during 10 min. Recombinant DNA ligases from P. abyssi and M. thermoautotrophicum possessed high stability during a storage at 4 degrees C.

Purification, crystallization and preliminary crystallographic analysis of a multiple cofactor-dependent DNA ligase from Sulfophobococcus zilligii.

A recombinant DNA ligase from Sulfophobococcus zilligii that shows multiple cofactor specificity (ATP, ADP and GTP) was expressed in Escherichia coli and purified under reducing conditions. Crystals were obtained by the microbatch crystallization method at 295 K in a drop containing 1 µl protein solution (10 mg ml(-1)) and an equal volume of mother liquor [0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 10 000]. A data set was collected to 2.9 Šresolution using synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a=63.7, b=77.1, c=77.8 Å, α=83.4, ß=82.4, γ=74.6°. Assuming the presence of two molecules in the unit cell, the solvent content was estimated to be about 53.4%.

XLF regulates filament architecture of the XRCC4·ligase IV complex.

DNA ligase IV (LigIV) is critical for nonhomologous end joining (NHEJ), the major DNA double-strand break (DSB) repair pathway in human cells, and LigIV activity is regulated by XRCC4 and XLF (XRCC4-like factor) interactions. Here, we employ small angle X-ray scattering (SAXS) data to characterize three-dimensional arrangements in solution for full-length XRCC4, XRCC4 in complex with LigIV tandem BRCT domains and XLF, plus the XRCC4·XLF·BRCT2 complex. XRCC4 forms tetramers mediated through a head-to-head interface, and the XRCC4 C-terminal coiled-coil region folds back on itself to support this interaction. The interaction between XLF and XRCC4 is also mediated via head-to-head interactions. In the XLF·XRCC4·BRCT complex, alternating repeating units of XLF and XRCC4·BRCT place the BRCT domain on one side of the filament. Collective results identify XRCC4 and XLF filaments suitable to align DNA molecules and function to facilitate LigIV end joining required for DSB repair in vivo.

Knockout of the DNA ligase IV homolog gene in the sphingoid base producing yeast Pichia ciferrii significantly increases gene targeting efficiency.

The yeast Pichia ciferrii produces large quantities of the sphingoid base tetraacetyl phytosphingosine (TAPS) and is an interesting platform organism for the biotechnological production of sphingolipids and ceramides. Ceramides have attracted great attention as a specialty ingredient for moisture retention and protection of the skin in the cosmetics industry. First attempts have been started to metabolically engineer P. ciferrii for improved production of TAPS and other sphingoid bases. However, rational metabolic engineering of P. ciferrii is difficult due to a low gene targeting efficiency. In eukaryotes, two major pathways coexist, which are responsible for genomic DNA integration, homologous recombination (HR) and non-homologous end joining (NHEJ). Integration via HR is targeted, while NHEJ involves ectopic (non-targeted) integration depending on a ligation step mediated by DNA ligase IV (Lig4). Here, we demonstrate a dramatical increase in gene targeting efficiency in a P. ciferrii lig4 knockout strain, deficient in NHEJ. Furthermore, a quick and easy to use freeze-thaw method was developed to transform P. ciferrii with high efficiency. Owing to the ability of targeting genomic DNA integration our results pave the way for further genetic and metabolic engineering approaches with P. ciferrii by means of knocking out or overexpressing predestinated genes.

ATP-dependent DNA ligase from Archaeoglobus fulgidus displays a tightly closed conformation.

DNA ligases join the breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 3'-hydroxyl and 5'-phosphate termini. They fall into two classes that require either ATP or NAD(+) as the source of an AMP group that is covalently attached to a strictly conserved lysine. Conformational flexibility is essential for the function of multi-domain DNA ligases because they must undergo large conformational changes involving domain rearrangements during the course of the reaction. In the absence of the nicked DNA substrate, both open and closed conformations have been observed for the ATP-dependent DNA ligases from Sulfolobus solfataricus and Pyrococcus furiosus. Here, the crystal structure of an ATP-dependent DNA ligase from Archaeoglobus fulgidus has been determined in the DNA-unbound unadenylated state. It resembles the closed conformation of P. furiosus DNA ligase but was even more closed, thus enhancing our understanding of the conformational variability of these enzymes.

Overexpression, purification and crystallization of a thermostable DNA ligase from the archaeon Thermococcus sp. 1519.

DNA ligases catalyze the sealing of 5'-phosphate and 3'-hydroxyl termini at single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome in DNA metabolism. An ATP-dependent DNA ligase from the archaeon Thermococcus sp. 1519 was overexpressed, purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion method employing 35%(v/v) Tacsimate pH 7.0 as a precipitant and diffracted X-rays to 3.09 A resolution. They belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 79.7, c = 182.6 A.

[Isolation and characteristics of new thermostable DNA ligase from archaea of the genus Thermococcus].

The DNA ligase gene from thermophilic archaea of the genus Thermococcus (strain 1519) was identified and sequenced in the polymerase chain reaction. The recombinant enzyme LigTh1519 was expressed in Escherichia coli, purified, and characterized. LigTh1519 was capable of ligating the cohesive ends and single-strand breaks in double-stranded DNA (ATP as a cofactor). The optimum conditions for the ligase reaction appeared as follows: 100 mM NaCl, 50 mM MgCl2, pH 7.0-10.5, and temperature 70 degrees C. More than 50% Lig1519 activity were preserved after incubation of the enzyme at 80 degrees C for 30 min. New thermostable DNA ligase LihTh1519 may be used for basic and applied researches in molecular biology and genetic engineering.

Identification and characterization of an inhibitor specific to bacterial NAD+-dependent DNA ligases.

DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require NAD(+) for activity, but eukaryotic and viral DNA ligases require ATP. Because of their essential nature, unique structures and widespread existence in nature, bacterial DNA ligases represent a class of valuable targets for identifying novel and selective antibacterial agents. In this study, we cloned and expressed the ligA gene from Streptococcus pneumoniae, and characterized this ligA-encoded NAD(+)-dependent DNA ligase. We then screened small molecule chemical libraries using a biochemical assay and identified a new small molecule with a structure of 2,4-diamino-7-dimethylamino-pyrimido[4,5-d]pyrimidine. We show that this small molecule is a specific inhibitor of bacterial NAD(+)-dependent DNA ligases. Biochemical studies show that this molecule inhibits NAD(+)-dependent DNA ligases, but not ATP-dependent enzymes. The molecule inhibits NAD(+)-dependent DNA ligases competitively with respect to NAD(+) and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD(+)-dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.

Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme.

The gene encoding Staphylothermus marinus DNA ligase (Sma DNA ligase) was cloned and sequenced. The gene contains an open reading frame consisting of 1836bp, which encodes for 611 amino acid residues. Upon alignment of the entire amino acid sequence, Sma DNA ligase showed a high degree of sequence homology with the hyperthemophilic archaeal DNA ligases, 67% identity with Aeropyrum pernix K1, and 40% identity with both Pyrococcus abyssi and Thermococcus kodakarensis. An extremely high sequence identity was observed in the six conserved motifs indicative of DNA ligase. The Sma DNA ligase gene was expressed under the control of the T7lac promoter on the pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was then purified by heat treatment followed by ion exchange and metal affinity column chromatography. The enzyme was activated by both Mg(2+) and Mn(2+), and its activity was inhibited by Ca(2+) and Zn(2+). Sma DNA ligase can utilize both ATP and ADP as cofactors. The half-life of the enzyme at 100 degrees C was determined to be approximately 2.8h. The enzyme catalyzed cohesive-end intramolecular and intermolecular joining and blunt-end intermolecular joining in the presence of tricine-NaOH buffer and Mn(2+), using either ATP or ADP.