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Archaea (Vancouver, B.C.) 2015With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and... (Review)
Review
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.
Topics: Archaea; Biotechnology; DNA Ligases; Protein Engineering; RNA Ligase (ATP)
PubMed: 26494982
DOI: 10.1155/2015/170571 -
International Journal of Molecular... Jan 2021X-ray analysis cannot provide quantitative estimates of the relative contribution of non-specific, specific, strong, and weak contacts of extended DNA molecules to their... (Review)
Review
X-ray analysis cannot provide quantitative estimates of the relative contribution of non-specific, specific, strong, and weak contacts of extended DNA molecules to their total affinity for enzymes and proteins. The interaction of different enzymes and proteins with long DNA and RNA at the quantitative molecular level can be successfully analyzed using the method of the stepwise increase in ligand complexity (SILC). The present review summarizes the data on stepwise increase in ligand complexity (SILC) analysis of nucleic acid recognition by various enzymes-replication, restriction, integration, topoisomerization, six different repair enzymes (uracil DNA glycosylase, Fpg protein from , human 8-oxoguanine-DNA glycosylase, human apurinic/apyrimidinic endonuclease, RecA protein, and DNA-ligase), and five DNA-recognizing proteins (RNA helicase, human lactoferrin, alfa-lactalbumin, human blood albumin, and IgGs against DNA). The relative contributions of structural elements of DNA fragments "covered" by globules of enzymes and proteins to the total affinity of DNA have been evaluated. Thermodynamic and catalytic factors providing discrimination of unspecific and specific DNAs by these enzymes on the stages of primary complex formation following changes in enzymes and DNAs or RNAs conformations and direct processing of the catalysis of the reactions were found. General regularities of recognition of nucleic acid by DNA-dependent enzymes, proteins, and antibodies were established.
Topics: Animals; Antibodies; DNA; DNA Glycosylases; DNA Helicases; DNA Ligases; DNA Topoisomerases, Type I; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Directed DNA Polymerase; Deoxyribonuclease EcoRI; Humans; Lactalbumin; Lactoferrin; Proteins; Rec A Recombinases; Serum Albumin, Human
PubMed: 33573045
DOI: 10.3390/ijms22031369 -
Journal of Molecular Biology May 2019DNA ligases are a highly conserved group of nucleic acid enzymes that play an essential role in DNA repair, replication, and recombination. This review focuses on... (Review)
Review
DNA ligases are a highly conserved group of nucleic acid enzymes that play an essential role in DNA repair, replication, and recombination. This review focuses on functional interaction between DNA polymerases and DNA ligases in the repair of single- and double-strand DNA breaks, and discusses the notion that the substrate channeling during DNA polymerase-mediated nucleotide insertion coupled to DNA ligation could be a mechanism to minimize the release of potentially mutagenic repair intermediates. Evidence suggesting that DNA ligases are essential for cell viability includes the fact that defects or insufficiency in DNA ligase are casually linked to genome instability. In the future, it may be possible to develop small molecule inhibitors of mammalian DNA ligases and/or their functional protein partners that potentiate the effects of chemotherapeutic compounds and improve cancer treatment outcomes.
Topics: Animals; DNA; DNA Breaks; DNA Ligases; DNA Repair; DNA-Directed DNA Polymerase; Genomic Instability; Humans; Protein Interaction Maps
PubMed: 31034893
DOI: 10.1016/j.jmb.2019.04.028 -
Nature Communications Nov 2019DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1...
DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3'-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. Here, X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA unveil that LIG1 employs Mg-reinforced DNA binding to validate DNA base pairing during the adenylyl transfer and nick-sealing ligation reaction steps. Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg, and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage.
Topics: DNA; DNA Breaks, Single-Stranded; DNA Damage; DNA Ligase ATP; DNA Repair; DNA Replication; DNA-Binding Proteins; Guanine; Humans; Magnesium; Nuclear Proteins; Nucleic Acid Conformation; Oxidation-Reduction; Protein Structure, Tertiary; Recombinational DNA Repair
PubMed: 31780661
DOI: 10.1038/s41467-019-13478-7 -
Journal of Thermal Biology Jul 2022Wood frogs, Rana sylvatica, endure the freezing of ∼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also...
Wood frogs, Rana sylvatica, endure the freezing of ∼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also long-term anoxia due to cessation of heartbeat and breathing. Thawing restores perfusion but rapid reoxygenation can increase vulnerability to reactive oxygen species and induce oxidative damage. This study provides a first assessment of antioxidant capacity, DNA damage, and DNA repair responses comparing freeze/thaw and anoxia/reoxygenation in liver and skeletal muscle of wood frogs. Oxidation of guanine resides in DNA did not change under either stress but total antioxidant capacity rose in both tissues under anoxia. Relative expression of eight proteins involved in double-stranded break repair (Mre11, Rad50, phospho-p95, XLF, DNA ligase IV, XRCC4, Ku70, Rad51) were assessed in both tissues. Freezing suppressed Ku70 and Rad51 in liver and Rad51 in muscle but levels rose again after thawing. Anoxia exposure suppressed XLF, Ku70 and Rad51 proteins in muscle. However, in liver, anoxia exposure led to elevated Mre11, Ku70 and DNA ligase IV, the former two belonging to the MRN complex that binds DNA and marks sites of double stranded breaks (DSBs). Large increases in Mre11 and Ku70 expression suggested DSB damage in liver under anoxia but not during freezing, whereas muscle was resistant to DSB damage under both stresses. These data indicate that DNA damage is minimal during whole body freezing due to tissue and stress specific regulation of antioxidant capacity and DNA damage repair to preserve genomic integrity.
Topics: Animals; Antioxidants; DNA; DNA Damage; DNA Ligase ATP; Freezing; Hypoxia; Muscle, Skeletal; Ranidae
PubMed: 35701025
DOI: 10.1016/j.jtherbio.2022.103274 -
Nature Structural & Molecular Biology Oct 2023DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the...
DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol IIIα, Pol I polymerase, Pol I endonuclease and DNA ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA-DNA junction, thus marking the end of the RNA primer and creating a 5' end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.
Topics: DNA Polymerase III; DNA; DNA Replication; RNA; DNA Ligases; DNA Ligase ATP; Endonucleases
PubMed: 37620586
DOI: 10.1038/s41594-023-01071-y -
Current Medicinal Chemistry 2019Antimicrobial resistance is found in all microorganisms and has become one of the biggest threats to global health. New antimicrobials with different action mechanisms... (Review)
Review
BACKGROUND
Antimicrobial resistance is found in all microorganisms and has become one of the biggest threats to global health. New antimicrobials with different action mechanisms are effective weapons to fight against antibiotic-resistance.
OBJECTIVE
This review aims to find potential drugs which can be further developed into clinic practice and provide clues for developing more effective antimicrobials.
METHODS
DNA replication universally exists in all living organisms and is a complicated process in which multiple enzymes are involved in. Enzymes in bacterial DNA replication of initiation and elongation phases bring abundant targets for antimicrobial development as they are conserved and indispensable. In this review, enzyme inhibitors of DNA helicase, DNA primase, topoisomerases, DNA polymerase and DNA ligase were discussed. Special attentions were paid to structures, activities and action modes of these enzyme inhibitors.
RESULTS
Among these enzymes, type II topoisomerase is the most validated target with abundant inhibitors. For type II topoisomerase inhibitors (excluding quinolones), NBTIs and benzimidazole urea derivatives are the most promising inhibitors because of their good antimicrobial activity and physicochemical properties. Simultaneously, DNA gyrase targeted drugs are particularly attractive in the treatment of tuberculosis as DNA gyrase is the sole type II topoisomerase in Mycobacterium tuberculosis. Relatively, exploitation of antimicrobial inhibitors of the other DNA replication enzymes are primeval, in which inhibitors of topo III are even blank so far.
CONCLUSION
This review demonstrates that inhibitors of DNA replication enzymes are abundant, diverse and promising, many of which can be developed into antimicrobials to deal with antibioticresistance.
Topics: Anti-Bacterial Agents; Bacteria; DNA Helicases; DNA Ligases; DNA Primase; Drug Resistance, Microbial; Humans; Nucleic Acid Synthesis Inhibitors; Topoisomerase II Inhibitors
PubMed: 29110590
DOI: 10.2174/0929867324666171106160326 -
DNA Repair Nov 2015DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base... (Review)
Review
DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER.
Topics: 8-Hydroxy-2'-Deoxyguanosine; DNA Damage; DNA Ligases; DNA Polymerase beta; DNA Repair; Deoxyguanosine; Genomic Instability; Hazardous Substances; Humans; Oxidants
PubMed: 26466358
DOI: 10.1016/j.dnarep.2015.09.010 -
Current Pharmaceutical Design 2019Base excision DNA repair (BER) is a vitally important pathway that protects the cell genome from many kinds of DNA damage, including oxidation, deamination, and... (Review)
Review
Base excision DNA repair (BER) is a vitally important pathway that protects the cell genome from many kinds of DNA damage, including oxidation, deamination, and hydrolysis. It involves several tightly coordinated steps, starting from damaged base excision and followed by nicking one DNA strand, incorporating an undamaged nucleotide, and DNA ligation. Deficiencies in BER are often embryonic lethal or cause morbid diseases such as cancer, neurodegeneration, or severe immune pathologies. Starting from the early 1980s, when the first mammalian cell lines lacking BER were produced by spontaneous mutagenesis, such lines have become a treasure trove of valuable information about the mechanisms of BER, often revealing unexpected connections with other cellular processes, such as antibody maturation or epigenetic demethylation. In addition, these cell lines have found an increasing use in genotoxicity testing, where they provide increased sensitivity and representativity to cell-based assay panels. In this review, we outline current knowledge about BER-deficient cell lines and their use.
Topics: Animals; Cell Line; DNA; DNA Damage; DNA Glycosylases; DNA Ligases; DNA Repair; DNA-Directed DNA Polymerase; Endonucleases; Humans
PubMed: 31198112
DOI: 10.2174/1381612825666190319112930 -
Nucleic Acids Research Jan 2023Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III...
Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3'-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5'-overhang-containing DNA substrates ∼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg2+, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg2+ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.
Topics: Animals; Humans; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Repair; Magnesium; Mammals
PubMed: 36625284
DOI: 10.1093/nar/gkac1263