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

Several DNA-interactive proteins, including the DNA repair enzyme T4 endonuclease V, have been shown to locate their target recognition sites utilizing an electrostatically mediated facilitated diffusion mechanism. Previous work indicates that a decrease in the affinity of endonuclease V for nontarget DNA results in an increased nontarget dissociation rate. This study was designed to investigate the effect of an increase in the affinity of endonuclease V for nontarget DNA. Using a working structural model of the enzyme as a guide, the electrostatic character of endonuclease V was altered. Substitution of Thr-7 with Lys-7 resulted in an enzyme with wild type in vitro characteristics. Mutations which increased the positive charge along a proposed solvent-exposed alpha-helical face had significant effects. The mutants Ala-30, Val-31----Lys-30, Leu-31 and Asn-37----Lys-37 displayed wild type in vitro apurinic-specific and dimer-specific nicking activities. Although the processive dimer-specific nicking rate of the Lys-37 mutant resembled that of wild type, the rate of the Lys-30, Leu-31 mutant was reduced by 60%. In addition, the salt concentration range over which these mutants processively nick dimer-containing DNA has been greatly expanded. Both mutants are shown to have an increased affinity for nontarget DNA.
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PMID:Substitution of basic amino acids within endonuclease V enhances nontarget DNA binding. 200 4

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.
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PMID:Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding. 203 8

The nucleotide sequence of 42090 bp of vaccinia virus strain WR is presented. The sequence includes the SalI L, F, G and I fragments and starts near the centre of the HindIII A fragment and extends rightwards towards the genomic terminus, finishing approximately 0.5 kb internal of the inverted terminal repeat (ITR). Translation of this region has identified 65 open reading frames (ORFs) of greater than 65 amino acids in length. Fifty-one of these which do not extensively overlap other larger ORFs have been subjected to further analysis; the other 14 are termed minor ORFs. In the rightmost 28.7 kb, the genes are, with one exception, transcribed towards the genomic terminus, similar to the arrangement of genes at the left end of the virus genome. Internal of this region the genes are expressed off either DNA strand but still predominately rightwards. ORFs are tightly packed with few intergenic non-coding regions of greater than 250 bp. Protein sequence comparisons have established a remarkably high number of homologies with entries in existing protein databases. Of these, DNA ligase, thymidylate kinase, two serine-threonine protein kinases, two serine proteinase inhibitors (serpins), two interleukin-1 receptor homologous and a discontinuous ORF related to tumour necrosis factor receptor have been reported. Other homologies include lectins, profilin, 3 beta-hydroxy steroid dehydrogenase, superoxide dismutase, guanylate kinase, ankyrin and complement factor H. In addition, there are a number of polypeptides with predicted properties of membrane-associated, secretory or glyco-proteins. Twelve gene families are described here and elsewhere. There is considerable similarity between genes from the right and left end of the virus genome that may have arisen by terminal transposition events. Several differences from the corresponding region of vaccinia virus strain Copenhagen sequence are noted. Near the right terminus the sequences diverge completely, and internal of this there are multiple examples of deletion of short sequences (eight to 10 nucleotides) that lie within penta- or hexanucleotide direct repeats.
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PMID:Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat. 204 93

Our recent structure-activity analysis of Fpg protein of Escherichia coli, using oligodeoxynucleotides containing various 8-oxopurine derivatives, has allowed us to postulate an enzyme mechanism involving protonation of 8-oxoguanine at O-6 and nucleophilic attack of the deoxyribose moiety at C-1' leading to the formation of an enzyme-substrate Schiff base intermediate (Tchou, J., Bodepudi, V., Shibutani, S., Antoshechkin, I., Miller, J., Grollman, A. P., and Johnson, F. (1994) J. Biol. Chem. 269, 15318-15324). In this paper, sodium cyanoborohydride has been used to convert the transient intermediate to a covalent enzyme-DNA complex. The location of the active site of Fpg protein is further delineated using two approaches. 1) A radiolabeled DNA substrate is used to tag the active site of Fpg protein, using sodium cyanoborohydride. The active site is mapped to the first 73 amino acid residue fragment by cyanogen bromide cleavage analysis. 2) A maltose-binding protein fusion system is used to generate amino-terminal modifications of Fpg protein to explore the role of the amino-terminal region in DNA binding and catalysis. Results support the conclusion that the active site of Fpg protein is located at or near the amino terminus. Thus, Fpg protein may act in a similar fashion as T4 endonuclease V, a DNA repair enzyme that uses its amino-terminal alpha-amino group of threonine to carry out catalysis via Schiff base formation (Dodson et al., 1993).
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PMID:The catalytic mechanism of Fpg protein. Evidence for a Schiff base intermediate and amino terminus localization of the catalytic site. 774 6

Angiotensin II (Ang II) type 2 (AT2) receptors are highly expressed in neonate brain and may have a role in developmental processes such as apoptosis. Concurrent activation of c-Jun N-terminal kinase (JNK) and inhibition of Erk mitogen-activated protein kinase activities is important for apoptosis in many cells, and we previously demonstrated that stimulation of AT2 receptors causes decreased mitogen-activated protein kinase activity in neurons cultured from newborn rat hypothalamus and brain stem. Using such cultures we have employed terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling and internucleosomal DNA fragmentation to assess the role of AT2 receptors in neuronal apoptosis. Ang II (100 nM; 4-72 h) alone produced no significant neuronal apoptosis, and AT2 receptor activation did not stimulate JNK activity. However, exposure of cultures to UV radiation (6 J/m2/sec for 4 sec) to stimulate JNK elicited neuronal apoptosis that was significantly enhanced by Ang II, an effect that was abolished by the AT2 receptor antagonist PD 123,319 (1 microM) or the serine/threonine phosphatase inhibitor okadaic acid (3 nM). Additionally, Ang II enhanced the UV radiation-induced decrease in the levels of the DNA repair enzyme poly-(ADP-ribose) polymerase. These data indicate that Ang II, via AT2 receptors and activation of a serine/threonine phosphatase, contributes to neuronal apoptosis.
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PMID:Angiotensin II type 2 receptor-mediated apoptosis of cultured neurons from newborn rat brain. 988 63

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.
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PMID:Role of nucleotidyl transferase motif V in strand joining by chlorella virus DNA ligase. 1175 16

Non-homologous end joining (NHEJ) is one of the primary pathways for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in mammalian cells. Proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku, XRCC4 and DNA ligase IV. Current models predict that DNA-PKcs, Ku, XRCC4 and DNA ligase IV assemble at DSBs and that the protein kinase activity of DNA-PKcs is essential for NHEJ-mediated repair of DSBs in vivo. We previously identified a cluster of autophosphorylation sites between amino acids 2609 and 2647 of DNA-PKcs. Cells expressing DNA-PKcs in which these autophosphorylation sites have been mutated to alanine are highly radiosensitive and defective in their ability to repair DSBs in the context of extrachromosomal assays. Here, we show that cells expressing DNA-PKcs with mutated autophosphorylation sites are also defective in the repair of IR-induced DSBs in the context of chromatin. Purified DNA-PKcs proteins containing serine/threonine to alanine or aspartate mutations at this cluster of autophosphorylation sites were indistinguishable from wild-type (wt) protein with respect to protein kinase activity. However, mutant DNA-PKcs proteins were defective relative to wt DNA-PKcs with respect to their ability to support T4 DNA ligase-mediated intermolecular ligation of DNA ends. We propose that autophosphorylation of DNA-PKcs at this cluster of sites is important for remodeling of DNA-PK complexes at DNA ends prior to DNA end joining.
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PMID:Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends. 1531 5

The response of eukaryotic cells to DNA damage requires a multitude of protein-protein interactions that mediate the ordered repair of the damage and the arrest of the cell cycle until repair is complete. Two conserved protein modules, BRCT and forkhead-associated (FHA) domains, play key roles in the DNA-damage response as recognition elements for nuclear Ser/Thr phosphorylation induced by DNA-damage-responsive kinases. BRCT domains, first identified at the C-terminus of BRCA1, often occur as multiple tandem repeats of individual BRCT modules. Our recent structural and functional work has revealed how BRCT repeats recognize phosphoserine protein targets. It has also revealed a secondary binding pocket at the interface between tandem repeats, which recognizes the amino-acid 3 residues C-terminal to the phosphoserine. We have also studied the molecular function of the FHA domain of the DNA repair enzyme, polynucleotide kinase (PNK). This domain interacts with threonine-phosphorylated XRCC1 and XRCC4, proteins responsible for the recruitment of PNK to sites of DNA-strand-break repair. Our studies have revealed a flexible mode of recognition that allows PNK to interact with numerous negatively charged substrates.
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PMID:Structural basis for phosphorylation-dependent signaling in the DNA-damage response. 1633 23

DNA polymerase (Pol) lambda is a DNA repair enzyme involved in base excision repair, non-homologous end joining and translesion synthesis. Recently, we identified Pol lambda as an interaction partner of cyclin-dependent kinase 2 (CDK2) that is central to the cell cycle G1/S transition and S-phase progression. This interaction leads to in vitro phosphorylation of Pol lambda, and its in vivo phosphorylation pattern during cell cycle progression mimics the modulation of CDK2/cyclin A. Here, we identify several phosphorylation sites of Pol lambda. Experiments with phosphorylation-defective mutants suggest that phosphorylation of Thr 553 is important for maintaining Pol lambda stability, as it is targeted to the proteasomal degradation pathway through ubiquitination unless this residue is phosphorylated. In particular, Pol lambda is stabilized during cell cycle progression in the late S and G2 phases. This most likely allows Pol lambda to correctly conduct repair of damaged DNA during and after S phase.
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PMID:Control of DNA polymerase lambda stability by phosphorylation and ubiquitination during the cell cycle. 1868 54

DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.
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PMID:DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories. 1959 47


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