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

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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)

Using synthetic oligodeoxynucleotides with 3'-OH ends and 32P-labelled 5'-phosphate ends and the technique of polyacrylamide gel electrophoresis, it is shown that, in the presence of the complementary polynucleotide, an AP (apurinic or apyrimidinic) site at the 3' or the 5' end of the labelled oligodeoxynucleotides does not prevent their ligation by T4 DNA ligase, although the reaction rate is decreased. This decrease is more severe when the AP site is at the 3' end; the activated intermediates accumulate showing that it is the efficiency of the adenyl-5'-phosphate attack by the 3'-OH of the base-free deoxyribose which is mostly perturbed. Using the same technique, it is shown that a mispaired base at the 3' or 5' end of oligodeoxynucleotides does not prevent their ligation. A one-nucleotide gap, limited by 3'-OH and 5'-phosphate, can also be closed by T4 DNA ligase although with difficulty; here again the activation of the 5'-phosphate end does not seem to be slowed down, but rather the 3'-OH attack of the adenyl-5'-phosphate. All these anomalous ligations take place with the nick or the gap in front of a continuous complementary strand. Blunt ends ligation of correct duplexes occurs readily; however an AP site or a mispaired base at the 3' or 5' end of one strand of the duplexes prevents ligation between these strands. But a missing nucleotide (responsible for one unpaired nucleotide protruding at the 3' or 5' end of the complementary strand) does not stop ligation of the shorter oligodeoxynucleotides between independent duplexes.
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PMID:Nicks 3' or 5' to AP sites or to mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase. 368 72

The 5' AP endodeoxyribonucleases hydrolyze the phosphodiester bond 5' to AP (apurinic or apyrimidinic) sites in double-stranded DNA leaving 3'-OH and 5'-phosphate ends. These nicks are sealed by T4 DNA ligase although the 5'-phosphate end belongs to a base-free deoxyribose.
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PMID:T4 DNA ligase can seal a nick in double-stranded DNA limited by a 5'-phosphorylated base-free deoxyribose residue. 622 66

The early steps of excision repair of cyclobutane pyrimidine dimers are investigated. It is demonstrated that the apurinic/apyrimidinic endonuclease associated with the Micrococcus luteus uv-specific endonuclease cleaves the phosphodiester bond on the 3' side of the deoxyribose leaving a 3' hydroxy terminus and a 5' phosphoryl terminus. This nick is not a substrate for T4 polynucleotide ligase. The 3' base-free deoxyribose terminus is not a substrate for either the polymerase or the 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I. However, the 3' terminus of the nick is converted to a substrate for DNA polymerization by the action of a 5' apurinic/apyrimidinic endonuclease. A three-step model for the incision step of excision repair of cyclobutane pyrimidine dimers is presented.
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PMID:Early steps of excision repair of cyclobutane pyrimidine dimers by the Micrococcus luteus endonuclease. A three-step incision model. 626 31

DNA repair proficiency in cells is expressed by various enzymes which can recognize damaged sites arising from exogenous agents or endogenous conditions. Either a damaged base is recognized by DNA glycosylases, partially removed by hemi-DNA glycosylases acting on diadduct damage, or direct incision of the phosphodiester bond near the damaged site. Incision at those apurinic or apyrimidinic sites arising from depurination-depyrimidination or glycosylase reactions is effected by apurinic or apyrimidinic endonucleases. Excision of damaged sites is catalyzed by unique exonucleases followed by DNA polymerase catalyzed reinsertion of nucleotides. The integrity of the strands is restored by polynucleotide ligase when a juxtaposed nucleotide is properly reinserted.
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PMID:Enzymatic mechanisms of DNA repair. 628 49

The ability of HeLa DNA polymerase alpha to utilize gapped PM2 DNAs for synthesis in a model base excision DNA repair scheme was examined. Partially depurinated PM2 DNA was incised on the 5' side of apurinic sites with HeLa apurinic/apyrimidinic endonuclease II, then the baseless sugar was removed and gaps of defined mean lengths were introduced at these sites by exonucleolytic digestion with HeLa DNase V. Gaps smaller than approximately 15 nucleotides did not serve as efficient primer-templates for DNA polymerase alpha. Gaps with mean lengths of 20-63 nucleotides did support limited DNA synthesis, but such synthesis terminated after the gap was reduced to roughly 15 nucleotides. These products were not substrates for Escherichia coli DNA ligase. In contrast, HeLa DNA polymerase beta utilize as primer-templates all of the gapped DNA substrates tested though it acted more efficiently with the smaller gaps. Moreover, the beta-polymerase was capable of filling these gaps to completion. In the case of the gaps that remained after partial closure by DNA polymerase alpha, DNA polymerase beta incorporated roughly 15 nucleotides and formed a product which was a substrate for DNA ligase. These results suggest that in vivo DNA repair pathways that involve a gap-filling DNA synthesis reaction might utilize DNA polymerase alpha only for larger gaps.
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PMID:Gap-filling DNA synthesis by HeLa DNA polymerase alpha in an in vitro base excision DNA repair scheme. 646 63

Essentially all of the DNA polymerase alpha activity in CV-1 monkey cells could be extracted as an enzyme complex that used DNA substrates with a low primer:template ratio, such as denatured DNA, at least 25 times more efficiently than did purified alpha polymerase. This form of the enzyme was rapidly dissociated either by the nonionic detergent Triton X-100 or by chromatography on phosphocellulose to generate alpha polymerase and its protein cofactor complex, C1C2. Both alpha polymerase and C1C2 were then independently purified free of deoxyribonuclease, RNA polymerase, DNA ligase, and ATPase activities, and the C1C2 complex was shown to consist of at least two proteins. Purified C1C2, which exhibited no DNA polymerase activity, completely restored the ability of alpha polymerase to use denatured DNA. Although high concentrations of denatured DNA inhibited the activity of C1C2, which binds tightly to single-stranded but not double-stranded DNA, low concentrations catalyzed reconstitution of alpha polymerase with C1C2. The resulting enzyme complex was chromatographically distinct from alpha polymerase on DEAE-Bio-Gel, was no longer dependent upon addition of C1C2 in order to utilize denatured DNA as effectively as DNase I-activated DNA, and was not inhibited by high concentrations of denatured DNA. These properties of the purified reconstituted enzyme were indistinguishable from those native alpha X C1C2-polymerase.
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PMID:Preparation of DNA polymerase alpha X C1C2 by reconstituting DNA polymerase alpha with its specific stimulatory cofactors, C1C2. 688 71

Ferric nitrilotriacetate (Fe(3+)-NTA) catalyzes hydrogen peroxide-derived production of hydroxyl radicals, which are known to cause DNA damage. In the present work, Fe(3+)-NTA plus hydrogen peroxide-induced single-strand DNA breaks and repair of the DNA damage were studied in vitro by monitoring DNA damage- and DNA repair-dependent conformational changes of pUC18 plasmid DNA. Single-strand DNA breaks were induced in the pUC18 DNA by Fe(3+)-NTA plus hydrogen peroxide in a dose-dependent fashion. Induction of the DNA damage was inhibited by deferoxamine mesylate (an iron chelator) and by hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO), D-mannitol and ethanol indicating that the DNA damage was caused by hydroxyl radicals which were generated by reaction of Fe(3+)-NTA with hydrogen peroxide. The oxygen radical-induced single-strand DNA breaks were repaired partly (more than 50%) by incubating the damaged DNA at 37 degrees C for 3 h with a partially purified preparation of APEX nuclease (a multifunctional DNA repair enzyme), DNA polymerase beta, four deoxyribonucleoside triphosphates, T4 DNA ligase and ATP. Analyses of the partially purified preparation of APEX nuclease revealed that a 45-kDa protein as well as APEX nuclease in the preparation were involved in the repair of the single-strand DNA breaks. APEX nuclease was suggested to initiate the repair by removing 3' termini blocked by the nucleotide fragments and also by incising the 5' side of AP sites. The 45-kDa protein was suggested to be required for removal of the 5' tags such as 5'-terminal deoxyribose phosphate residues produced by the action of APEX nuclease on AP sites.
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PMID:Oxygen radical-induced single-strand DNA breaks and repair of the damage in a cell-free system. 756 64

Ref-1 is a nuclear protein that possesses DNA repair activity and has a role in the redox activation of Fos and Jun transcription factors. Using an antibody to Ref-1 we investigated the expression and distribution of this protein in the adult rat brain. Ref-1 was located in the nucleus of neurons and glial fibrillary acidic protein-positive astrocytes throughout the brain. Levels were particularly high in granule cells of the dentate gyrus, piriform cortex neurons, and Purkinje cells of the cerebellum, and lower in CA1 pyramidal cells, striatal neurons, and the neurons of the neocortex. These results suggest that the action of inducible transcription factors such as c-Jun in mammalian neurons is likely to be regulated by constitutively expressed Ref-1, in particular in dentate granule cells. The high levels of Ref-1 in glial fibrillary acidic protein-positive astrocytes suggest that it may also modulate the action of inducible transcription factors in these cells, particularly after brain injury. The possibility also exists that Ref-1 may primarily function as a DNA repair enzyme in brain cells.
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PMID:Ref-1 expression in adult mammalian neurons and astrocytes. 764 43

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.
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PMID:DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. 782 35

APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. This report describes the organization of the gene (APEX gene) for human APEX nuclease. Human APEX gene was cloned using human APEX cDNA and a human leukocyte genomic library in bacteriophage vector EMBL-3. We proved that human APEX gene consists of 5 exons spanning 2.64 kilobases and suggested that the gene exists as a single copy in the haploid genome. The boundaries between exon and intron follow the GT/AG rule. The major transcription initiation site was assigned by primer extension analysis to C at 515 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites locate in the exon II and V, respectively. The 5' flanking region (0.89 kilobase) sequenced lacks typical TATA and CAAT boxes, but contains TATA- and CAAT-like sequences and putative cis-acting regulatory elements such as binding sites for Sp1, AP2 and ATF. A part of the 5' flanking region belongs to a CpG island, which extends to the intron II. The CpG island is thought to be a transcription regulatory region of APEX gene, a housekeeping gene. The promoter activity of the 5' upstream region was analyzed by introducing the region in HeLa cells in an expression construct containing luciferase gene as a reporter gene, and the region from position 130 bp upstream to position 205 bp downstream of the major transcription initiation site was shown to be enough for high promoter activity. Northern hybridization experiments suggested that the gene is expressed ubiquitously in human cells. The locus of APEX gene was mapped to human chromosome 14q11.2-q12 using the in situ hybridization technique.
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PMID:Structure, promoter analysis and chromosomal assignment of the human APEX gene. 808 53


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