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Nucleic Acids Research Oct 1999Repair synthesis catalysed by DNA polymerase beta at 1 nt gaps occurs in the main pathway of mammalian base excision repair. DNA polymerase beta has no exonucleolytic...
Repair synthesis catalysed by DNA polymerase beta at 1 nt gaps occurs in the main pathway of mammalian base excision repair. DNA polymerase beta has no exonucleolytic proof-reading ability, and exhibits high error frequency during DNA synthesis. Consequently, continuous correction of endogenous DNA damage by short-patch repair synthesis might lead to a high spontaneous mutation rate, unless subsequent steps in the repair pathway allow for selective removal of incorporation errors. We show here that both human DNA ligase I and III discriminate strongly between a correctly paired versus a mispaired residue at the 3' position of a nick in DNA, when assayed in the presence of physiological concentrations of KCl. The resulting delay in joining after misincorporation by DNA polymerase beta during gap filling could allow for removal of the mismatched terminal residue by a distinct 3' exonuclease.
Topics: Base Sequence; Catalysis; DNA Ligase ATP; DNA Ligases; DNA Repair; Humans; Kinetics; Molecular Sequence Data; Poly-ADP-Ribose Binding Proteins; Potassium Chloride; Substrate Specificity; Xenopus Proteins
PubMed: 10497267
DOI: 10.1093/nar/27.20.4028 -
PloS One 2017Escherichia coli encodes two DNA ligases, ligase A, which is essential under normal laboratory growth conditions, and ligase B, which is not. Here we report potential...
Escherichia coli encodes two DNA ligases, ligase A, which is essential under normal laboratory growth conditions, and ligase B, which is not. Here we report potential functions of ligase B. We found that across the entire Enterobacteriaceae family, ligase B is highly conserved in both amino acid identity and synteny with genes associated with oxidative stress. Deletion of ligB sensitized E. coli to specific DNA damaging agents and antibiotics resulted in a weak mutator phenotype, and decreased biofilm formation. Overexpression of ligB caused a dramatic extension of lag phase that eventually resumed normal growth. The ligase function of ligase B was not required to mediate the extended lag phase, as overexpression of a ligase-deficient ligB mutant also blocked growth. Overexpression of ligB during logarithmic growth caused an immediate block of cell growth and DNA replication, and death of about half of cells. These data support a potential role for ligase B in the base excision repair pathway or the mismatch repair pathway.
Topics: DNA Damage; DNA Ligases; DNA Replication; Enterobacteriaceae; Escherichia coli; Oxidative Stress
PubMed: 28700629
DOI: 10.1371/journal.pone.0180800 -
The Journal of Biological Chemistry Nov 2000Repair of DNA double-strand breaks in mammalian cells occurs via a direct nonhomologous end-joining pathway. Although this pathway can be studied in vivo and in crude...
Repair of DNA double-strand breaks in mammalian cells occurs via a direct nonhomologous end-joining pathway. Although this pathway can be studied in vivo and in crude cell-free systems, a deeper understanding of the mechanism requires reconstitution with purified enzymes. We have expressed and purified a complex of two proteins that are critical for double-strand break repair, DNA ligase IV (DNL IV) and XRCC4. The complex is homogeneous, with a molecular mass of about 300,000 Da, suggestive of a mixed tetramer containing two copies of each polypeptide. The presence of multiple copies of DNL IV was confirmed in an experiment where different epitope-tagged forms of DNL IV were recovered simultaneously in the same complex. Cross-linking suggests that an XRCC4.XRCC4 dimer interface forms the core of the tetramer, and that the DNL IV polypeptides are in contact with XRCC4 but not with one another. Purified DNL IV.XRCC4 complex functioned synergistically with Ku protein, the DNA-dependent protein kinase catalytic subunit, and other repair factors in a cell-free end-joining assay. We suggest that a dyad-symmetric DNL IV.XRCC4 tetramer bridges the two ends of the broken DNA and catalyzes the coordinate ligation of the two DNA strands.
Topics: Animals; Base Sequence; Biopolymers; Cell-Free System; DNA Ligase ATP; DNA Ligases; DNA Primers; DNA-Binding Proteins; HeLa Cells; Humans; Molecular Weight; Precipitin Tests; Spodoptera
PubMed: 10945980
DOI: 10.1074/jbc.M004011200 -
Journal of Genetics Aug 2014Elucidation of the genetic diversity and relatedness of the subpopulations of India may provide a unique resource for future analysis of genetic association of several...
Elucidation of the genetic diversity and relatedness of the subpopulations of India may provide a unique resource for future analysis of genetic association of several critical community-specific complex diseases.We performed a comprehensive exploration of single nucleotide polymorphisms (SNPs) within the gene DNA ligase 1 (LIG1) among a multiethnic panel of Indian subpopulations representative of the ethnic, linguistic and geographical diversity of India using a two-stage design involving DNA resequencing-based SNP discovery followed by SNP validation using sequenom-based genotyping. Thirty SNPs were identified in LIG1 gene using DNA resequencing including three promoter SNPs and one coding SNP. Following SNP validation, the SNPs rs20580/C19008A and rs3730862/C8804T were found to have the most widespread prevalence with noticeable variations in minor allele frequencies both between the Indian subpopulation groups and also from those reported on other major world populations. Subsequently, SNPs found in Indian subpopulations were analysed using bioinformatics-based approaches and compared with SNP data available on major world populations. Further, we also performed genotype-phenotype association analysis of LIG1 SNPs with publicly available data on LIG1 mRNA expression in HapMap samples. Results showed polymorphisms in LIG1 affect its expression and may therefore change its function. Our results stress upon the uniqueness of the Indian population with respect to the worldwide scenario and suggest that any epidemiological study undertaken on the global population should take this distinctiveness in consideration and avoid making generalized conclusions.
Topics: DNA Ligase ATP; DNA Ligases; Gene Frequency; Genetic Association Studies; HapMap Project; Haplotypes; Humans; India; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Sequence Analysis, DNA
PubMed: 25189241
DOI: 10.1007/s12041-014-0415-7 -
Scientific Reports Dec 2015With the advent of nanotechnology, a variety of nanoarchitectures with varied physicochemical properties have been designed. Owing to the unique characteristics, DNAs...
With the advent of nanotechnology, a variety of nanoarchitectures with varied physicochemical properties have been designed. Owing to the unique characteristics, DNAs have been used as a functional building block for novel nanoarchitecture. In particular, a self-assembly of long DNA molecules via a piece DNA staple has been utilized to attain such constructs. However, it needs many talented prerequisites (e.g., complicated computer program) with fewer yields of products. In addition, it has many limitations to overcome: for instance, (i) thermal instability under moderate environments and (ii) restraint in size caused by the restricted length of scaffold strands. Alternatively, the enzymatic sewing linkage of short DNA blocks is simply designed into long DNA assemblies but it is more error-prone due to the undeveloped sequence data. Here, we present, for the first time, a comprehensive study for directly combining DNA structures into higher DNA sewing constructs through the 5'-end cohesive ligation of T4 enzyme. Inspired by these achievements, the synthesized DNA nanomaterials were also utilized for effective detection and real-time diagnosis of cancer-specific and cytosolic RNA markers. This generalized protocol for generic DNA sewing is expected to be useful in several DNA nanotechnology as well as any nucleic acid-related fields.
Topics: Biosensing Techniques; DNA; DNA Ligases; Humans; Nanostructures; Nanotechnology; Nucleic Acid Conformation; RNA
PubMed: 26634810
DOI: 10.1038/srep17722 -
Journal of Biochemistry Mar 2004DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first...
DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase. The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme. The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2 mM ATP could inhibit its activity by 50%. Under optimal conditions (pH 8.0, 0.5 mM ATP, 5 mM DTT, 1 mM Mg(2+) and 300 mM Na(+)), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30 min. T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for blunt-ended DNA fragments. Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.
Topics: Amino Acid Sequence; Bacteriophage T3; Cloning, Molecular; Coenzymes; DNA; DNA Ligases; Enzyme Stability; Hydrogen-Ion Concentration; Ions; Molecular Sequence Data; Nucleotides; Phylogeny; Sequence Alignment; Temperature
PubMed: 15113838
DOI: 10.1093/jb/mvh047 -
Molecular and Cellular Biology Aug 2003The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are...
The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.
Topics: Cell Nucleus; DNA Damage; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA-Binding Proteins; Deoxyribonuclease I; Glutathione Transferase; HeLa Cells; Humans; Immunoblotting; Mass Spectrometry; Plasmids; Poly(ADP-ribose) Polymerases; Poly-ADP-Ribose Binding Proteins; Precipitin Tests; Protein Structure, Tertiary; Recombinant Fusion Proteins; Subcellular Fractions; Surface Plasmon Resonance; Time Factors; Two-Hybrid System Techniques; X-ray Repair Cross Complementing Protein 1; Xenopus Proteins
PubMed: 12897160
DOI: 10.1128/MCB.23.16.5919-5927.2003 -
The Journal of Biological Chemistry Jun 2013Nonhomologous end joining repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. Ku, XRCC4/Ligase IV (XL), and XLF have a remarkable...
Nonhomologous end joining repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. Ku, XRCC4/Ligase IV (XL), and XLF have a remarkable mismatched end (MEnd) ligase activity, particularly for ends with mismatched 3' overhangs, but the mechanism has remained obscure. Here, we showed XL required Ku to bind DNA, whereas XLF required both Ku and XL to bind DNA. We detected cooperative assembly of one or two Ku molecules and up to five molecules each of XL and XLF into a Ku-XL-XLF-DNA (MEnd ligase-DNA) complex. XLF mutations that disrupted its interactions with XRCC4 or DNA also disrupted complex assembly and end joining. Together with published co-crystal structures of truncated XRCC4 and XLF proteins, our data with full-length Ku, XL, and XLF bound to DNA indicate assembly of a filament containing Ku plus alternating XL and XLF molecules. By contrast, in the absence of XLF, we detected cooperative assembly of up to six molecules each of Ku and XL into a Ku-XL-DNA complex, consistent with a filament containing alternating Ku and XL molecules. Despite a lower molecular mass, MEnd ligase-DNA had a lower electrophoretic mobility than Ku-XL-DNA. The anomalous difference in mobility and difference in XL to Ku molar ratio suggests that MEnd ligase-DNA has a distinct structure that successfully aligns mismatched DNA ends for ligation.
Topics: Antigens, Nuclear; DNA; DNA End-Joining Repair; DNA Ligase ATP; DNA Ligases; DNA Repair Enzymes; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Electrophoretic Mobility Shift Assay; Humans; Ku Autoantigen; Models, Genetic; Mutation
PubMed: 23620595
DOI: 10.1074/jbc.M113.464115 -
Virology Sep 2006Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication,...
Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication, modification, and repair. Here we produced, purified, and characterized mimivirus DNA ligase (MimiLIG), an NAD+-dependent nick joining enzyme homologous to bacterial LigA and entomopoxvirus DNA ligase. MimiLIG is a 636-aa polypeptide composed of an N-terminal NAD+ specificity module (domain Ia), linked to nucleotidyltransferase, OB-fold, helix-hairpin-helix, and BRCT domains, but it lacks the tetracysteine Zn-binding module found in all bacterial LigA enzymes. MimiLIG requires conserved domain Ia residues Tyr36, Asp46, Tyr49, and Asp50 for its initial reaction with NAD+ to form the ligase-AMP intermediate, but not for the third step of phosphodiester formation at a preadenylylated nick. MimiLIG differs from bacterial LigA enzymes in that its activity is strongly dependent on the C-terminal BRCT domain, deletion of which reduced its specific activity in nick joining by 75-fold without affecting the ligase adenylylation step. The DeltaBRCT mutant of MimiLIG was impaired in sealing at a preadenylylated nick. We propose that eukaryal DNA viruses acquired the NAD+-dependent ligases by horizontal transfer from a bacterium and that MimiLIG predates entomopoxvirus ligase, which lacks both the tetracysteine and BRCT domains. We speculate that the dissemination of NAD+-dependent ligase from bacterium to eukaryotic virus might have occurred within an amoebal host.
Topics: Acanthamoeba; Amino Acid Motifs; Amino Acid Sequence; Animals; Conserved Sequence; Cysteine; DNA Ligases; Entomopoxvirinae; Molecular Sequence Data; Protein Structure, Tertiary; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 16844179
DOI: 10.1016/j.virol.2006.04.032 -
The Biochemical Journal Jan 2015DNA replication on the lagging strand occurs via the synthesis and maturation of Okazaki fragments. In archaea and eukaryotes, the enzymatic activities required for this...
DNA replication on the lagging strand occurs via the synthesis and maturation of Okazaki fragments. In archaea and eukaryotes, the enzymatic activities required for this process are supplied by a replicative DNA polymerase, Flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). These factors interact with the sliding clamp PCNA (proliferating cell nuclear antigen) providing a potential means of co-ordinating their sequential actions within a higher order assembly. In hyperthermophilic archaea of the Sulfolobus genus, PCNA is a defined heterotrimeric assembly and each subunit interacts preferentially with specific client proteins. We have exploited this inherent asymmetry to assemble a PCNA-polymerase-Fen1-ligase complex on DNA and have visualized it by electron microscopy. Our studies reveal the structural basis of co-occupancy of a single PCNA ring by the three distinct client proteins.
Topics: Archaeal Proteins; DNA Ligase ATP; DNA Ligases; Flap Endonucleases; Multiprotein Complexes; Proliferating Cell Nuclear Antigen; Sulfolobus solfataricus
PubMed: 25299633
DOI: 10.1042/BJ20141120