Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The release of reactive oxygen species (ROS) has been proposed as a cause of streptozotocin (STZ)-induced beta-cell damage. This initiates a destructive cascade, consisting of DNA damage, excess activation of the DNA repair enzyme poly(ADP-ribose) polymerase, and depletion of cellular NAD+. Metallothionein (MT) is an inducible antioxidant protein that has been shown to protect DNA from chemical damage in several cell types. Therefore, we examined whether overexpression of MT could protect beta-cell DNA and thereby prevent STZ-induced diabetes. Two lines of transgenic mice were produced with up to a 30-fold elevation in beta-cell MT. Cultured islets from control mice and MT transgenic mice were exposed to STZ. MT was found to decrease STZ-induced islet disruption, DNA breakage, and depletion of NAD+. To assess in vivo protection, transgenic and control mice were injected with STZ. Transgenic mice had significantly reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated mice showed that MT prevented degranulation and cell death. These results demonstrate that MT can reduce diabetes and confirm the DNA damage mechanism of STZ-induced beta-cell death.
...
PMID:Overexpression of metallothionein in pancreatic beta-cells reduces streptozotocin-induced DNA damage and diabetes. 1152 69

ATP-dependent DNA ligases, NAD(+)-dependent DNA ligases, and GTP-dependent RNA capping enzymes are members of a covalent nucleotidyl transferase superfamily defined by a common fold and a set of conserved peptide motifs. Here we examined the role of nucleotidyl transferase motif V ((184)LLKMKQFKDAEAT(196)) in the nick joining reaction of Chlorella virus DNA ligase, an exemplary ATP-dependent enzyme. We found that alanine substitutions at Lys(186), Lys(188), Asp(192), and Glu(194) reduced ligase specific activity by at least an order of magnitude, whereas substitutions at Lys(191) and Thr(196) were benign. The K186A, D192A, and E194A changes had no effect on the rate of single-turnover nick joining by preformed ligase-adenylate but affected subsequent rounds of nick joining at the ligase adenylation step. Conservative substitutions K186R, D192E, and E194D partially restored activity, whereas K186Q, D192N, and E194Q substitutions did not. Alanine mutation of Lys(188) elicited distinctive catalytic defects, whereby single-turnover nick joining by K188A-adenylate was slowed by an order of magnitude, and high levels of the DNA-adenylate intermediate accumulated. The rate of phosphodiester bond formation at a pre-adenylated nick (step 3 of the ligation pathway) was slowed by the K188A change. Replacement of Lys(188) by arginine reversed the step 3 arrest, whereas glutamine substitution was ineffective. Gel-shift analysis showed that the Lys(188) mutants bound stably to DNA-adenylate. We infer that Lys(188) is involved in the chemical step of phosphodiester bond formation.
...
PMID:Role of nucleotidyl transferase motif V in strand joining by chlorella virus DNA ligase. 1175 16

The adaptive response is an error-free DNA repair mechanism induced by low levels of physical or chemical agents. Cells pre-exposed to such agents are resistant to genetic damage induced by subsequent treatment at a high dose. There are many reports on such adaptive responses. Recently we have shown the existence of adaptive responses in vivo in the grasshopper Poecilocerus pictus and the mouse and in vitro in human lymphocytes. Different enzymes are implicated in this DNA repair pathway. In an attempt to understand the molecular mechanism of the methyl methanesulfonate (MMS)-induced adaptive response, the present investigations have been undertaken employing nicotinamide, an inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP). Pre-, inter- and post-treatments with nicotinamide of MMS-treated mouse bone marrow cells were carried out. The results revealed that there is a significant reduction in the frequency of chromosomal aberrations compared with combined treatment, suggesting an enhancement of the adaptive response by nicotinamide. Further, the results of NAD+ assay in the inter-treatment experiment showed that there is no depletion of NAD+. Thus, it can be stated that PARP is not involved in the MMS-induced adaptive response in mouse bone marrow cells.
...
PMID:Inducible protective processes in animal systems. X. Influence of nicotinamide in methyl methanesulfonate-adapted mouse bone marrow cells. 1175 27

NAD(+)-dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD(+) ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD(+) and then to isolate small molecules that recognize these components and thereby block the binding of NAD(+) to the ligase. The limitation to this strategy is that the structural determinants of NAD(+) specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD(+) requires N-terminal domain Ia, which is unique to, and conserved among, NAD(+) ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD(+). Structure-activity relationships were clarified by conservative substitutions.
...
PMID:Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+. 1178 21

Escherichia coli DNA ligase (LigA) is the prototype of the NAD(+)-dependent class of DNA ligases found in all bacteria. Here we report the characterization of E.coli LigB, a second NAD(+)-dependent DNA ligase identified by virtue of its sequence similarity to LigA. LigB differs from LigA in that it lacks the BRCA1 C-terminus domain (BRCT) and two of the four Zn-binding cysteines that are present in LigA and all other bacterial NAD(+) ligases. We found that recombinant LigB catalyzed strand joining on a singly-nicked DNA in the presence of a divalent cation and NAD(+), and that LigB reacted with NAD(+) to form a covalent ligase-adenylate intermediate. Alanine substitution for the motif I lysine ((126)KxDG) abolished nick joining and ligase-adenylate formation by LigB, thus confirming that the ligase and adenylyltransferase activities are intrinsic to the LigB protein.
...
PMID:A second NAD(+)-dependent DNA ligase (LigB) in Escherichia coli. 1181 21

Toxic reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide, and the hydroxyl radical are generated in a variety of neuropathological conditions and cause significant DNA damage. We determined the effects of 3-aminobenzamide (AB), an inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), on cell death in differentiated PC12 cells, a model of sympathetic neurons, after H(2) O(2) injury. Exposure to 0.5 mm H(2) O(2) resulted in a significant decrease in intracellular NAD(H), NADP(H), and ATP levels. This injury resulted in the death of 90% of the cells with significant necrosis early (2 h) after injury and increased apoptosis (12-24 h after injury), as measured by PS exposure and the presence of cytoplasmic oligonucleosomal fragments. Treatment with 2.5 mm AB restored pyridine nucleotide and ATP levels and ameliorated cell death (65% versus 90%) by decreasing the extent of both necrosis and apoptosis. Interestingly, we observed that H(2) O(2) -induced injury caused a delayed cell death exhibiting features of apoptosis but in which caspase-3 like activity was absent. Moreover, pretreatment with AB restored caspase-3-like activity. Our results suggest that apoptosis and necrosis are both triggered by PARP overactivation, and that maintenance of cellular energy levels after injury by inhibiting PARP shifts cell death from necrosis to apoptosis.
...
PMID:Poly(ADP-ribose) polymerase inhibition prevents both apoptotic-like delayed neuronal death and necrosis after H(2)O(2) injury. 1209 61

The DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP1), contributes to cell death during ischemia/reperfusion when extensively activated by DNA damage. The cell death resulting from PARP1 activation is linked to NAD+ depletion and energy failure, but the intervening steps are not well understood. Because glycolysis requires cytosolic NAD+, the authors tested whether PARP1 activation impairs glycolytic flux and whether substrates that bypass glycolysis can rescue cells after PARP1 activation. PARP1 was activated in mouse cortical astrocyte and astrocyte-neuron cocultures with the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Studies using the 2-deoxyglucose method confirmed that glycolytic flux was reduced by more than 90% in MNNG-treated cultures. The addition of 5 mmol/L of alpha-ketoglutarate, 5 mmol/L pyruvate, or other mitochondrial substrates to the cultures after MNNG treatment reduced cell death from approximately 70% to near basal levels, while PARP inhibitors and excess glucose had negligible effects. The mitochondrial substrates significantly reduced cell death, with delivery delayed up to 2 hours after MNNG washout. The findings suggest that impaired glycolytic flux is an important factor contributing to PARP1-mediated cell death. Delivery of alternative substrates may be a promising strategy for delayed treatment of PARP1-mediated cell death in ischemia and other disorders.
...
PMID:Tricarboxylic acid cycle substrates prevent PARP-mediated death of neurons and astrocytes. 1214 62

Previous studies have shown that the liver is the first organ to display signs of injury during hemorrhagic shock. We examined the mechanism by which pyruvate can prevent liver damage during hemorrhagic shock in swine anesthetized with halothane. Thirty minutes after the induction of a 240-min controlled arterial hemorrhage targeted at 40 mmHg, hypertonic sodium pyruvate (0.5 g. kg(-1). h(-1)) was infused to achieve an arterial concentration of 5 mM. The volume and osmolality effects of pyruvate were matched with 10% saline (HTS) and 0.9% saline (NS). Although the peak hemorrhage volume increased significantly in both the pyruvate and HTS group, only the pyruvate treatment was effective in delaying cardiovascular decompensation. In addition, pyruvate effectively maintained the NADH/NAD redox state, as evidenced by increased microdialysate pyruvate levels and a significantly lower lactate-to-pyruvate ratio. Pyruvate also prevented the loss of intracellular antioxidants (GSH) and a reduction in the GSH-to-GSSG ratio. These beneficial effects on the redox environment decreased hepatic cellular death by apoptosis. Pyruvate significantly increased the ratio of Bcl-Xl (antiapoptotic molecule)/Bax (proapoptotic molecule), prevented the release of cytochrome c from mitochondria, and decreased the fragmentation of caspase 3 and poly(ADP ribose) polymerase (DNA repair enzyme). These beneficial findings indicate that pyruvate infused 30 min after the onset of severe hemorrhagic shock is effective in maintaining the redox environment, preventing the loss of the key antioxidant GSH, and decreasing early apoptosis indicators.
...
PMID:Pyruvate improves redox status and decreases indicators of hepatic apoptosis during hemorrhagic shock in swine. 1223 18

DNA ligase is an enzyme essential for DNA replication, repair, and recombination in all organisms. Bacterial DNA ligases catalyze a NAD(+)-dependent DNA ligation reaction, i.e., the formation of a phosphodiester bond between adjacent 3'-OH and 5'-phosphate termini of dsDNA. Due to their essential nature, unique cofactor requirement, and widespread existence in nature, bacterial DNA ligases appear to be valuable targets for identifying novel antibacterial agents. To explore bacterial DNA ligases as antibacterial targets and further characterize them, we developed a simple, robust, homogeneous time-resolved fluorescence resonance energy transfer assay (TR-FRET) for measuring Streptococcus pneumoniae DNA ligase activity. This assay involves the use of one dsDNA molecule labeled with biotin and another dsDNA molecule labeled with Cy5, an acceptor fluorophore. During ligation reactions, the donor fluorophore europium (Eu(3+)) labeled with streptavidin was added to the assay mixtures, which bound to the biotin label on the ligated products. This in turn resulted in the FRET from Eu(3+) to Cy5 due to their close proximity. The formation of ligation products was measured by monitoring the emission at 665nm. This assay was validated by the experiments showing that the DNA ligase activity required NAD(+) and MgCl(2), and was inhibited by NMN and AMP, products of the ligase reaction. Using this assay, we determined the K(m) values of the enzyme for dsDNA substrates and NAD(+), and the IC(50) values of NMN and AMP, examined the effects of MgCl(2) and PEG(8000) on the enzyme activity, optimized the concentrations of Eu(3+) in the assay, and validated its utilities for high-throughput screening and biochemical characterizations of this class of enzymes.
...
PMID:Development of a fluorescence resonance energy transfer assay for measuring the activity of Streptococcus pneumoniae DNA ligase, an enzyme essential for DNA replication, repair, and recombination. 1241 56

A gene encoding a putative ATP-dependent DNA ligase was identified in the genome of the hyperthermophilic archaeon Sulfolobus shibatae and expressed in Escherichia coli. The 601 amino acid recombinant polypeptide was a monomeric protein capable of strand joining on a singly nicked DNA substrate in the presence of ATP ( K(m)=34 micro mu) and a divalent cation (Mn(2+), Mg(2+), or Ca(2+)). dATP was partially active in supporting ligation catalyzed by the protein, but GTP, CTP, UTP, dGTP, dCTP, dTTP, and NAD(+) were inactive. The cloned Ssh ligase showed an unusual metal cofactor requirement; it was significantly more active in the presence of Mn(2+) than in the presence of Mg(2+) or Ca(2+). Unexpectedly, the native Ssh ligase preferred Mg(2+) and Ca(2+) rather than Mn(2+). Both native and recombinant enzymes displayed optimal nick-joining activity at 60-80 degrees C. Ssh ligase discriminated against substrates containing mismatches on the 3'-side of nick junction and was more tolerant of mismatches at the 5'-end than of those at the penultimate 5'-end. The enzyme showed little activity on a 1-nucleotide gapped substrate. This is the first biochemical study of a DNA ligase from the crenarchaeotal branch of the archaea domain.
...
PMID:Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon Sulfolobus shibatae. 1248 55


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---