Single-strand Breaks In Double-stranded DNA Research Articles

ATP-dependent DNA ligase from Thermococcus sp. 1519 displays a new arrangement of the OB-fold domain.

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 for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 Å shows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.

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.

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.

The Closed Structure of an Archaeal DNA Ligase from Pyrococcus furiosus

DNA ligases join single-strand breaks in double-stranded DNA, and are essential to maintain genome integrity in DNA metabolism. Here, we report the 1.8 Å resolution structure of Pyrococcus furiosus DNA ligase (PfuLig), which represents the first full-length atomic view of an ATP-dependent eukaryotic-type DNA ligase. The enzyme comprises the N-terminal DNA-binding domain, the middle adenylation domain, and the C-terminal OB-fold domain. The architecture of each domain resembles those of human DNA ligase I, but the domain arrangements differ strikingly between the two enzymes. The closed conformation of the two “catalytic core” domains at the carboxyl terminus in PfuLig creates a small compartment, which holds a non-covalently bound AMP molecule. This domain rearrangement results from the “domain-connecting” role of the helical extension conserved at the C termini in archaeal and eukaryotic DNA ligases. The DNA substrate in the human open-ligase is replaced by motif VI in the Pfu closed-ligase. Both the shapes and electrostatic distributions are similar between motif VI and the DNA substrate, suggesting that motif VI in the closed state mimics the incoming substrate DNA. Two basic residues (R531 and K534) in motif VI reside within the active site pocket and interact with the phosphate group of the bound AMP. The crystallographic and functional analyses of mutant enzymes revealed that these two residues within the RxDK sequence play essential and complementary roles in ATP processing. This sequence is also conserved exclusively among the covalent nucleotidyltransferases, even including mRNA-capping enzymes with similar helical extensions at the C termini.

Overexpression, purification and crystallization of an archaeal DNA ligase fromPyrococcus furiosus

DNA ligases seal single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome during various aspects of DNA metabolism, such as replication, excision repair and recombination. DNA-strand breaks are frequently generated as reaction intermediates in these events and the sealing of these breaks depends solely on the proper function of DNA ligase. Crystals of the archaeal DNA ligase from Pyrococcus furiosus were obtained using 6.6%(v/v) ethanol as a precipitant and diffracted X-rays to 1.7 A resolution. They belong to the monoclinic space group P2(1), with unit-cell parameters a = 61.1, b = 88.3, c = 63.4 A, beta = 108.9 degrees. The asymmetric unit contains one ligase molecule.

Resistance of 3'-phosphoglycolate DNA ends to digestion by mammalian DNase III.

An essential step in the repair of free radical-mediated DNA strand breaks is the removal of sugar fragments such as phosphoglycolate from the 3' termini. While the abasic endonuclease Ape1 can remove phosphoglycolate from single-strand breaks in double-stranded DNA, an enzyme capable of removing it from 3' overhangs of double-strand breaks has yet to be identified. We therefore tested DNase III, the predominant 3' --> 5' exonuclease in mammalian cell extracts, for possible 3'-phosphoglycolate-removing activity. However, all 3'-phosphoglycolate substrates, as well as a 3'-phosphate substrate, were resistant to DNase III under conditions in which the analogous 3'-hydroxyl substrates were extensively degraded. The DNA end-binding protein Ku (an equimolar mixture of Ku70, now known as G22P1, and Ku86, now known as XRCC5) did not alter the resistance of the 3'-phosphoglycolate substrates, but the protein modulated the susceptibility of 3'-hydroxyl substrates, allowing DNase III to remove a 3' overhang but inhibiting digestion of the double-stranded portion of the substrate.

Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a “constant ·OH radical scavenging environment”, k of 1.5 × 107 s-1 by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0 ± 0.2 at [F] < 100 μM by a mechanism other than through direct scavenging of ·OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H· atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from ·OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.

Single-Strand Breaks in Double-Stranded DNA Irradiated in Anoxic Solution: Contribution of tert-Butanol Radicals

Yields of single-strand breaks induced by 60Co gamma or pulse irradiation in double-stranded calf thymus DNA have been measured in N2O-saturated aqueous solution as a function of the concentration of tert-butanol. The yields were found to be dependent on dose rate. The experimental data were analyzed using a theoretical model based on non-homogeneous scavenging kinetics. It is concluded from this analysis that after 60Co gamma irradiation in the absence of oxygen, aside from breaks caused by hydroxyl radicals, additional breaks occur which are initiated by hydrogen atoms and secondary radicals of tert-butanol. The efficiency of hydrogen atoms in causing single-strand breaks in double-stranded calf thymus DNA was determined to be 2.3%, while the rate constant for the reaction of tert-butanol radicals with DNA and their efficiency in causing single-strand breaks was determined to be 4.1 x 10(3) dm3 mol-1 s-1 and 2%, respectively.

Yields of Single-Strand Breaks in Double-Stranded Calf Thymus DNA Irradiated in Aqueous Solution in the Presence of Oxygen and Scavengers

Yields of radiation-induced single-strand breaks in double-stranded calf thymus DNA have been measured as a function of OH. scavenger concentration in N2O/O2-saturated aqueous solution. The experimental data are well represented by a theoretical model based on non-homogeneous reaction kinetics, without the need to adjust any parameter. The good agreement between experimental and theoretical data is taken as evidence that, in the presence of oxygen, the main effect of added scavengers with respect to the formation of single-strand breaks in double-stranded DNA is OH. radical scavenging.

Accumulation of DNA fragments in aging Paramecium tetraurelia in axenic and nonaxenic media.

The accumulation of DNA fragments in aging Paramecium tetraurelia after 15 days (or approximately 60 generations) of clonal growth in both axenic and nonaxenic media was assayed by alkaline elution assay. This sensitive technique permits measurement of single-strand breaks in double-stranded DNA. The results obtained indicate that P. tetraurelia aged in axenic medium accumulate more apurinic/apyrimidinic lesions, strand breaks, or single-strand gaps than do P. tetraurelia aged in nonaxenic medium. This implies that cells grown in axenic medium have a shorter lifespan than do cells grown in nonaxenic medium. The accumulation of DNA fragments may be the basic mechanism of aging in P. tetraurelia.

27] Endonuclease IV from Escherichia coli

Publisher Summary This chapter presents an overview of endonuclease IV from Escherichia coli. Endonuclease IV specifically catalyzes the introduction of single-strand breaks at apurinic and apyrimidinic sites in double-stranded DNA. The assay of endonuclease IV is based on the release of acid-soluble material from heavily depurinated radioactive DNA. Purification procedure is further highlighted; Escherichia coli 1100 is grown in a glucose-salts medium supplemented with 0.2% casamino acids and 0.1% yeast extract and harvested in the logarithmic growth phase. The cell paste is frozen at -70 ° and stored at that temperature. All purification steps are performed at 0 ° to 4 °. All buffers contain 10 -3 M dithiothreitol. Centrifugations are at 20,000 g for 15 rain. Properties are reviewed, such as stability and purity. The purified enzyme has a molecular weight of 30,000-33,000 as estimated by gel filtration and sucrose gradient centrifugation. Endonuclease IV catalyzes the formation of single-strand breaks in double-stranded DNA containing apurinic sites, introduced, example, by pH 5 treatment, or apyrimidinic sites introduced by treatment of DNA containing dUMP residues with uracil-DNA glycosylase.

Action of the single-stranded DNA specific nuclease S 1 on double-stranded DNA

The single-stranded DNA specific nuclease S 1 (Ando, T. (1966) Biochim. Biophys. Acta 114, 158–168) puts single-stranded breaks into double-stranded ΦX174 and T7 DNAs. The enzyme first converts the supercoiled ΦX174 RF I ★ ★ The double-stranded replicative form of Φ174 DNA is abbreviated throughout this paper as RF I (closed supercoiled form) or RF II (open circular form). into the relaxed form RF II, and then puts an increasing number of nicks into both of the DNA strands of the duplex. These breaks can only be observed upon denaturation of the molecule. This double-stranded DNA nicking activity of S 1 nuclease is low compared with the single-stranded DNA digestion activity, and during the time it takes to completely digest ΦX174 single-stranded DNA to acid soluble nucleotides ( i.e. approx. 5000 phosphodiester bond cleavages), only two to three nicks are put into the double-stranded ΦX174 DNA molecule ( i.e. four to six phosphodiester bond cleavages). As the enzyme preparation used gave a single band on a sodium dodecyl sulphate polyacrylamide gel, it is tentatively concluded that the S 1 nuclease not only digests single-stranded DNA to nucleotides, but also puts single-strand breaks in double-stranded DNA.

DNA-strand breaks in two strains of Escherichia coli after exposure to -rays.

SummaryA progressive decrease in the sedimentation rate of the DNA from Escherichia coli, wild type (parent strain), and the radioresistant mutant 6γ, indicating single-strand breaks in double-stranded DNA, was obtained after exposure of the strains to an increasing dose of γ-rays. Sedimentation of the DNA from irradiated cells of E. coli 6γ was restored to the pre-irradiation rate after a period of post-irradiation growth. No such effect was obtained with irradiated cells of the parent strain. Amino-acid starvation or addition of chloramphenicol after irradiation inhibited the restoration of the sedimentation rate of DNA.