-
Nucleic Acids Research Nov 2018DNA ligases play essential roles in DNA replication and repair. Bacteriophage T4 DNA ligase is the first ATP-dependent ligase enzyme to be discovered and is widely used...
DNA ligases play essential roles in DNA replication and repair. Bacteriophage T4 DNA ligase is the first ATP-dependent ligase enzyme to be discovered and is widely used in molecular biology, but its structure remained unknown. Our crystal structure of T4 DNA ligase bound to DNA shows a compact α-helical DNA-binding domain (DBD), nucleotidyl-transferase (NTase) domain, and OB-fold domain, which together fully encircle DNA. The DBD of T4 DNA ligase exhibits remarkable structural homology to the core DNA-binding helices of the larger DBDs from eukaryotic and archaeal DNA ligases, but it lacks additional structural components required for protein interactions. T4 DNA ligase instead has a flexible loop insertion within the NTase domain, which binds tightly to the T4 sliding clamp gp45 in a novel α-helical PIP-box conformation. Thus, T4 DNA ligase represents a prototype of the larger eukaryotic and archaeal DNA ligases, with a uniquely evolved mode of protein interaction that may be important for efficient DNA replication.
Topics: Archaea; Crystallography, X-Ray; DNA; DNA Ligase ATP; DNA Ligases; DNA, Archaeal; Eukaryota; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Conformation, alpha-Helical; Protein Domains
PubMed: 30169742
DOI: 10.1093/nar/gky776 -
Cancer Research May 2008Based 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...
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.
Topics: Computer-Aided Design; DNA; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA Replication; Drug Design; Drug Screening Assays, Antitumor; Enzyme Inhibitors; HCT116 Cells; HeLa Cells; Humans; Models, Biological; Models, Molecular; Protein Binding; Tumor Cells, Cultured
PubMed: 18451142
DOI: 10.1158/0008-5472.CAN-07-6636 -
Frontiers in Cellular and Infection... 2018The protozoan parasite is exposed to reactive oxygen and nitric oxide species that have the potential to damage its genome. harbors enzymes involved in DNA repair...
The protozoan parasite is exposed to reactive oxygen and nitric oxide species that have the potential to damage its genome. harbors enzymes involved in DNA repair pathways like Base and Nucleotide Excision Repair. The majority of DNA repairs pathways converge in their final step in which a DNA ligase seals the DNA nicks. In contrast to other eukaryotes, the genome of encodes only one DNA ligase (EhDNAligI), suggesting that this ligase is involved in both DNA replication and DNA repair. Therefore, the aim of this work was to characterize EhDNAligI, its ligation fidelity and its ability to ligate opposite DNA mismatches and oxidative DNA lesions, and to study its expression changes and localization during and after recovery from UV and HO treatment. We found that EhDNAligI is a high-fidelity DNA ligase on canonical substrates and is able to discriminate erroneous base-pairing opposite DNA lesions. EhDNAligI expression decreases after DNA damage induced by UV and HO treatments, but it was upregulated during recovery time. Upon oxidative DNA damage, EhDNAligI relocates into the nucleus where it co-localizes with EhPCNA and the 8-oxoG adduct. The appearance and disappearance of 8-oxoG during and after both treatments suggest that DNA damaged was efficiently repaired because the mainly NER and BER components are expressed in this parasite and some of them were modulated after DNA insults. All these data disclose the relevance of EhDNAligI as a specialized and unique ligase in that may be involved in DNA repair of the 8-oxoG lesions.
Topics: DNA Damage; DNA Ligases; DNA Repair; Entamoeba histolytica
PubMed: 30050869
DOI: 10.3389/fcimb.2018.00214 -
Nucleic Acids Research Feb 2012DNA double-strand breaks pose a significant threat to cell survival and must be repaired. In higher eukaryotes, such damage is repaired efficiently by non-homologous end...
DNA double-strand breaks pose a significant threat to cell survival and must be repaired. In higher eukaryotes, such damage is repaired efficiently by non-homologous end joining (NHEJ). Within this pathway, XRCC4 and XLF fulfill key roles required for end joining. Using DNA-binding and -bridging assays, combined with direct visualization, we present evidence for how XRCC4-XLF complexes robustly bridge DNA molecules. This unanticipated, DNA Ligase IV-independent bridging activity by XRCC4-XLF suggests an early role for this complex during end joining, in addition to its more well-established later functions. Mutational analysis of the XRCC4-XLF C-terminal tail regions further identifies specialized functions in complex formation and interaction with DNA and DNA Ligase IV. Based on these data and the crystal structure of an extended protein filament of XRCC4-XLF at 3.94 Å, a model for XRCC4-XLF complex function in NHEJ is presented.
Topics: Binding Sites; DNA; DNA Ligase ATP; DNA Ligases; DNA Repair Enzymes; DNA-Binding Proteins; Humans; Microscopy, Atomic Force; Models, Molecular; Protein Binding
PubMed: 22287571
DOI: 10.1093/nar/gks022 -
Genes To Cells : Devoted To Molecular &... Feb 1999Recent progress over the past year has provided new insights into the proteins involved in nonhomologous end joining. The assembly of Ku and DNA-dependent protein kinase... (Review)
Review
Recent progress over the past year has provided new insights into the proteins involved in nonhomologous end joining. The assembly of Ku and DNA-dependent protein kinase at DNA ends is now understood in greater detail. Murine genetic knockouts for DNA ligase IV and XRCC4 are embryonic lethal, indicating that nonhomologous end joining is essential for viability. Interestingly, neurones, in addition to lymphocytes, are particularly vulnerable to an absence of NHEJ.
Topics: Aging; Animals; Antigens, Nuclear; DNA; DNA Helicases; DNA Ligase ATP; DNA Ligases; DNA-Activated Protein Kinase; DNA-Binding Proteins; Eukaryotic Cells; Humans; Ku Autoantigen; Mice; Nuclear Proteins; Protein Serine-Threonine Kinases
PubMed: 10320474
DOI: 10.1046/j.1365-2443.1999.00245.x -
Nucleic Acids Research Oct 1987A nuclear DNA ligase activity from immature chicken erythrocytes, and to a lesser extent T4-induced DNA ligase, can join cohesive-ends (3 and 5-nucleotides long) having...
A nuclear DNA ligase activity from immature chicken erythrocytes, and to a lesser extent T4-induced DNA ligase, can join cohesive-ends (3 and 5-nucleotides long) having one of the mismatches, A/A, T/T, C/C, G/G, at the middle position. The rate of ligation depends on the length and stability of the mispaired intermediate (G/G, T/T greater than A/A, C/C). When the non-complementary overhanging-ends are short (i.e. 1-nucleotide) both ligases catalyze the joining of the single-stranded protruding-end with a blunt-end. This reaction occurs at low but significant rates compared to blunt-end ligation. The chicken ligase has lower flush-end joining activity than T4 DNA ligase, but it is more permissive since it joins C/C or A/A mismatched-ends, whereas the prokaryotic ligase does not. Possible biological implications of the reactions are discussed. We have also found that BstEII easily cleaves at sites harboring a C/C or a G/G mismatch at the center of its recognition sequence, whereas AvaII (T/T or A/A), HinfI (G/G) and DdeI (G/G) do not.
Topics: Animals; Base Composition; Chickens; DNA; DNA Ligases; DNA Restriction Enzymes; Erythrocytes; Models, Biological; Polynucleotide Ligases; Thermodynamics; Viral Proteins
PubMed: 2823219
DOI: 10.1093/nar/15.19.7831 -
DNA Repair Jul 2006DNA ligase IV is an essential protein that functions in DNA non-homologous end-joining, the major mechanism that rejoins DNA double-strand breaks in mammalian cells....
DNA ligase IV is an essential protein that functions in DNA non-homologous end-joining, the major mechanism that rejoins DNA double-strand breaks in mammalian cells. LIG4 syndrome represents a human disorder caused by mutations in DNA ligase IV that lead to impaired but not ablated activity. Thus far, five conserved motifs in DNA ligases have been identified. We previously reported G469E as a mutational change in a LIG4 syndrome patient. G469 does not lie in any of the previously reported motifs. A sequence comparison between DNA ligases led us to identify residues 468-476 of DNA ligase IV as a further conserved motif, designated motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly, however, such mutational changes do impact upon double-stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggest that residues 470-476 function as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.
Topics: Adenosine Triphosphate; Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Animals; Binding Sites; Cell Line; DNA; DNA Ligase ATP; DNA Ligases; DNA Repair; Humans; In Vitro Techniques; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Protein Conformation; Recombinant Proteins; Spodoptera
PubMed: 16735143
DOI: 10.1016/j.dnarep.2006.03.011 -
Cold Spring Harbor Perspectives in... May 2013Nonhomologous end joining (NHEJ) refers to a set of genome maintenance pathways in which two DNA double-strand break (DSB) ends are (re)joined by apposition, processing,... (Review)
Review
Nonhomologous end joining (NHEJ) refers to a set of genome maintenance pathways in which two DNA double-strand break (DSB) ends are (re)joined by apposition, processing, and ligation without the use of extended homology to guide repair. Canonical NHEJ (c-NHEJ) is a well-defined pathway with clear roles in protecting the integrity of chromosomes when DSBs arise. Recent advances have revealed much about the identity, structure, and function of c-NHEJ proteins, but many questions exist regarding their concerted action in the context of chromatin. Alternative NHEJ (alt-NHEJ) refers to more recently described mechanism(s) that repair DSBs in less-efficient backup reactions. There is great interest in defining alt-NHEJ more precisely, including its regulation relative to c-NHEJ, in light of evidence that alt-NHEJ can execute chromosome rearrangements. Progress toward these goals is reviewed.
Topics: DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Ligases; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Deoxyribonucleases; Histones; Models, Genetic; Mutation
PubMed: 23637284
DOI: 10.1101/cshperspect.a012757 -
Cell Reports Apr 2014Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby...
Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.
Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA; DNA Ligase ATP; DNA Ligases; DNA Replication; Gene Deletion; Mice; Poly-ADP-Ribose Binding Proteins; Xenopus Proteins
PubMed: 24726358
DOI: 10.1016/j.celrep.2014.03.024 -
American Journal of Human Genetics May 1999
Review
Topics: Animals; Carbon-Oxygen Lyases; DNA Damage; DNA Ligases; DNA Polymerase gamma; DNA Repair; DNA, Mitochondrial; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Directed DNA Polymerase; Deoxyribonuclease IV (Phage T4-Induced); Humans; Vertebrates
PubMed: 10205257
DOI: 10.1086/302392