Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease. The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence. We have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity. Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis. In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein. Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity. Loss of ATPase activity was about 75% in the K37A, 86% in the K646A mutant, and 95% in the K37A-K646A double mutant. These amino acid substitutions had only a marginal effect on the specific binding of UvrA to damaged DNA but drastically reduced its ability to deliver UvrB to the damage site. We find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA)2(UvrB)1 complexes. We conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA.
 ...
PMID:Site-specific mutagenesis of conserved residues within Walker A and B sequences of Escherichia coli UvrA protein. 182 50

tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q). It catalyzes the posttranscriptional base exchange of the Q precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine in the anticodon of tRNA(Asp), tRNA(Asn), tRNA(His), and tRNA(Tyr). A partially sequenced gene upstream of the DNA ligase (lig) gene of the Zymomonas mobilis chromosome shows strong homology to the tgt gene of Escherichia coli (K.B. Shark and T. Conway, FEMS Microbiol. Lett. 96:19-26, 1992). We showed that this gene is able to complement the tgt mutation in E. coli SJ1505, and we determined its complete sequence. Four start codons were possible for this gene, resulting in proteins of 386 to 399 amino acids (M(r), 42,800 to 44,300) showing 60.4% sequence identity with Tgt from E. coli. The smallest of the four possible reading frames, which was still extended at its 5' end compared with the E. coli tgt gene, was overexpressed in E. coli. The gene product was purified to homogeneity and was biochemically characterized. The kinetical parameters were virtually identical to those published for the E. coli enzyme. In contrast to E. coli Tgt, which is reported to be a homotrimer, Z. mobilis Tgt was found to be a monomer according to gel filtration. In this study, it was shown that the formation of homotrimers by the E. coli enzyme is readily reversible and is dependent on protein concentration.
 ...
PMID:Sequence analysis and overexpression of the Zymomonas mobilis tgt gene encoding tRNA-guanine transglycosylase: purification and biochemical characterization of the enzyme. 766 16

Mammalian cells contain three biochemically distinct DNA ligases. In this report we describe the purification of DNA ligase II to homogeneity from bovine liver nuclei. This enzyme interacts with ATP to form an enzyme-AMP complex, in which the AMP moiety is covalently linked to a lysine residue. An adenylylated peptide from DNA ligase II contains the sequence, Lys-Tyr-Asp-Gly-Glu-Arg, which is homologous to the active site motif conserved in ATP-dependent DNA ligases. The sequences adjacent to this motif in DNA ligase II are different from the comparable sequences in DNA ligase I, demonstrating that these enzymes are encoded by separate genes. The amino acid sequences of 15 DNA ligase II peptides exhibit striking homology (65% overall identity) with vaccinia DNA ligase. These peptides are also homologous (31% overall identity) with the catalytic domain of mammalian DNA ligase I, indicating that the genes encoding DNA ligases I and II probably evolved from a common ancestral gene. Since vaccinia DNA ligase is not required for DNA replication but influences the ability of the virus to survive DNA damage, the homology between this enzyme and DNA ligase II suggests that DNA ligase II may be involved in DNA repair.
 ...
PMID:Mammalian DNA ligase II is highly homologous with vaccinia DNA ligase. Identification of the DNA ligase II active site for enzyme-adenylate formation. 798 68

Interleukin-1beta-converting enzyme (ICE)/Ced-3 proteases play a critical role in apoptosis. One well characterized substrate of these proteases is the DNA repair enzyme poly(ADP-ribose) polymerase. We report here that alpha-fodrin, an abundant membrane-associated cytoskeletal protein, is cleaved rapidly and specifically during Fas- and tumor necrosis factor-induced apoptosis; this cleavage is mediated by an ICE/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. Studies in cells treated with these apoptotic stimuli reveal that both fodrin and poly(ADP-ribose) polymerase proteolysis are inhibited by acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and CrmA, specific inhibitors of ICE/Ced-3 proteases. However, fodrin proteolysis can be distinguished from poly(ADP-ribose) polymerase proteolysis by its relative insensitivity to acetyl-Asp-Glu-Val-Asp aldehyde (DEVD-CHO), a selective inhibitor of a subset of ICE/Ced-3 proteases that includes CPP32. DEVD-CHO protects cells from Fas-induced apoptosis but does not prevent fodrin proteolysis, indicating that cleavage of this protein can be uncoupled from apoptotic cell death. Moreover, purified fodrin is cleaved in vitro by CPP32 (but not by ICE) into fragments of the same size observed in vivo during apoptosis. These findings suggest that fodrin proteolysis in vivo may reflect the activity of multiple ICE/Ced-3 proteases whose partial sensitivity to DEVD-CHO reflects a limited contribution from CPP32, or an ICE/Ced-3 protease less sensitive than CPP32 to DEVD-CHO inhibition.
 ...
PMID:Specific cleavage of alpha-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated by an interleukin-1beta-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. 894 Jan 32

Interleukin-1beta-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1beta (pre-IL-1beta) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P1 aspartate residue. We have examined the kinetics of processing pre-IL-1beta to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1beta, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 x 10(6) M-1 s-1 for CPP32 to 1.0 x 10(3) M-1 s-1 for TX. In addition, we report kinetic constants for several reversible inhibitors and irreversible inactivators, which have been used to implicate one or more caspases in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) is a potent inhibitor of CPP-32 with a Ki value of 0.5 nM, but is also potent as inhibitor of CMH-1 (Ki = 35 nM) and ICE (Ki = 15 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presented here reveals electrostatic interactions not present in the Ac-YVAD-CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275), accounting for the surprising potency of this inhibitor against ICE.
 ...
PMID:Substrate and inhibitor specificity of interleukin-1 beta-converting enzyme and related caspases. 905 18

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.
 ...
PMID:Chlorella virus DNA ligase: nick recognition and mutational analysis. 942 10

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.
 ...
PMID:Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells. 969 73

The gene encoding Thermus filiformis (Tfi) DNA ligase was cloned and its nucleotide sequence was determined by the chain-termination method. The primary structure of Tfi DNA ligase was deduced from its nucleotide sequence. The Tfi DNA ligase comprises of 667 amino acid residues and its molecular mass was determined to be 75,936 Da. The deduced amino acid sequence of Tfi DNA ligase showed a 86.5% homology to Tth DNA ligase and 43.5% to E. coli DNA ligase. The Lys-116 of Lys-Val-Asp-Gly motif was proposed to be the active residue of Tfi DNA ligase. In comparison with the amino acid composition of DNA ligase, Tfi DNA ligase showed a significant increase in the proportion of charged residues, Arg and Glu, compared to E. coli DNA ligase. The G + C content in the first, second, and third positions of the codons used were 70.3%, 40.3%, and 90.3%, respectively. Codon usage in Tfi DNA ligase was heavily biased towards the use of G + C in the third position. Under tac promoter control, Tfi DNA ligase was overproduced to greater than 9% of E. coli BL26Blue cellular proteins.
 ...
PMID:Cloning, nucleotide sequence, and expression of the DNA ligase-encoding gene from Thermus filiformis. 974 31

Chlorambucil and prednisolone, two commonly used drugs in the treatment of chronic lymphocytic leukemia (CLL), induce apoptosis in CLL cells. We have investigated the involvement in this apoptotic cell death of caspases, which cleave critical cellular substrates thereby acting as the executioners of the apoptotic process. Induction of spontaneous or chlorambucil/prednisolone-induced apoptosis of freshly isolated B-CLL cells in culture resulted in the activation of the 'effector' caspases, -3 and -7, but generally not of caspase-2. Activation of caspases-3 and -7 was accompanied by the proteolysis of the DNA repair enzyme, poly (ADP-ribose) polymerase. Induction of apoptosis was also accompanied by the processing of caspase-8, the extent of which varied between patients. Induction of apoptosis and processing of all the caspases was inhibited by the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.fmk). Our results demonstrate a key role for the activation and processing of caspases in the execution phase of apoptosis in CLL cells. Apoptosis of CLL cells resulted in the selective activation of some but not all caspases. Our results suggest that the dysregulation of apoptosis observed in CLL may be due to the signalling leading to the activation of caspases rather than a deletion of pro-caspases. High levels of caspase-8 in CLL cells in conjunction with low levels of CD95 receptor may offer new therapeutic opportunities for the treatment of CLL.
 ...
PMID:Processing/activation of caspases, -3 and -7 and -8 but not caspase-2, in the induction of apoptosis in B-chronic lymphocytic leukemia cells. 976 99

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.
 ...
PMID:Role of DNA-dependent protein kinase in neuronal survival. 1143 81


1 2 3 Next >>