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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas' disease. We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism. The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable. The gene is single copy and is located on a chromosome of approximately 1.9 Mb. A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existence of a single UDGase species in parasite extracts. Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA. The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity. This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.
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PMID:Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease. 1126 57

The present investigation describes the construction of a genetically engineered single chain antibody (scFv) against the rat transferrin receptor (OX26), and demonstrates that this scFv antibody can be fully processed and expressed as a soluble secreted molecule in the methylotrophic yeast Pichia pastoris. Restriction endonuclease sites located at both 5'- and 3'-flanking regions of OX26 coding region in the prokaryote pOPE-OX26 vector were engineered to incorporate yeast compatible restriction endonuclease sites (i.e. EcoRI and SmaI or AvrII). The modified OX26 cDNA was subcloned into the Pichia expression vectors pPIC9 and pHIL-S1. An OX26 scFv high producer clone [GS115 His+ Mut+ (pPIC-OX26 SacI)] was isolated and used for large-scale production and characterization. Because the engineered scFv contains both a c-myc tag and a (His)5 tail, the OX26 scFv was purified to homogeneity by immobilized metal affinity chromatography. The identity of the OX26 scFv was confirmed by Western blot analyses with both anti c-myc and anti poly-His antibodies. Minor immunoreactive bands corresponding to hyperglycosylated and partially processed alpha-factor leader prosequence were also detected in the purified OX26 scFv, and these contaminants were markedly reduced when the expression of the OX26 scFv was performed in minimal methanol medium buffered with phosphate at pH = 7. The present investigation suggests that this expression system may be useful for the production of anti-receptor single chain antibodies that can be used as brain drug delivery vectors.
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PMID:Cloning and expression in Pichia pastoris of a genetically engineered single chain antibody against the rat transferrin receptor. 1132 66

Caspase-activated DNase (CAD) is a deoxyribonuclease that causes DNA fragmentation during apoptosis. In proliferating cells, CAD is complexed with ICAD (inhibitor of CAD) and its DNase activity is suppressed. Here, we established a quantitative assay for CAD DNase that measures the number of 3' hydroxyl groups on the CAD-generated DNA fragments. Chemical modification of histidine residues and substrate protection experiments demonstrated the presence of reactive histidine residues within the active site of the enzyme. Analysis by site-directed mutagenesis suggested that at least four histidine residues in the C-terminal part of the molecule are essential for the catalytic activity of CAD DNase. ICAD did not protect CAD from the chemical modification of the histidine residues, indicating that it does not mask the active site of CAD. In contrast, ICAD blocked the ability of CAD to bind DNA, suggesting that ICAD causes steric or electrostatic hindrance in CAD for substrate DNA. This molecular mechanism for the inhibition of CAD DNase by ICAD is similar to that proposed for colicin endonuclease and its inhibitor, immunity protein.
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PMID:Enzymatic active site of caspase-activated DNase (CAD) and its inhibition by inhibitor of CAD. 1136 Nov 46

Chicken anemia virus (CAV) isolates show extremely limited genetic variability worldwide. We determined the nucleotide sequence of an 823-nucleotide portion of the 2.3-kb CAV genome found in 10 liver and/or spleen specimens of Alabama 29-to-49-day-old commercial broiler chickens exhibiting lymphocyte depletion of the thymus submitted to the state diagnostic laboratory because of problems unrelated to anemia. We determined the nucleotide sequence directly from DNA isolated from tissues, without isolation of virus in culture. This procedure enabled us to characterize CAV that might not have replicated in culture and avoided the potential for changes during passage. Results confirmed the limited genetic variability of CAV. All sequences were identical in 93% of nucleotide positions. The sequences encoded only two distinct VP1 hypervariable regions, and both had been found previously in other CAV isolates. A novel amino acid, glutamine, was found at VP1 position 22 in half the sequences, replacing the histidine residue encoded by most previously characterized CAV genomes. We were able to distinguish among CAV genomes with different codons at VP1 amino acid 22 and different hypervariable regions by restriction endonuclease analysis of polymerase chain reaction products.
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PMID:Genetic characterization of chicken anemia virus from commercial broiler chickens in Alabama. 1141 17

Our previous study of the North American biogeography of Bangia revealed the presence of two introns inserted at positions 516 and 1506 in the nuclear-encoded SSU rRNA gene. We subsequently sequenced nuclear SSU rRNA in additional representatives of this genus and the sister genus Porphyra in order to examine the distribution, phylogeny, and structural characteristics of these group I introns. The lengths of these introns varied considerably, ranging from 467 to 997 nt for intron 516 and from 509 to 1,082 nt for intron 1506. The larger introns contained large insertions in the P2 domain of intron 516 and the P1 domain of intron 1506 that correspond to open reading frames (ORFs) with His-Cys box homing endonuclease motifs. These ORFs were found on the complementary strand of the 1506 intron in Porphyra fucicola and P. umbilicalis (HG), unlike the 516 intron in P. abbottae, P. kanakaensis, P. tenera (SK), Bangia fuscopurpurea (Helgoland), and B. fuscopurpurea (MA). Frameshifts were noted in the ORFs of the 516 introns in P. kanakaensis and B. fuscopurpurea (HL), and all ORFs terminated prematurely relative to the amino acid sequence for the homing endonuclease I-Ppo I. This raises the possibility that these sequences are pseudogenes. Phylogenies generated using sequences of both introns and the 18S rRNA gene were congruent, which indicated long-term immobility and vertical inheritance of the introns followed by subsequent loss in more derived lineages. The introns within the florideophyte species Hildenbrandia rubra (position 1506) were included to determine relationships with those in the Bangiales. The two sequences of intron 1506 analyzed in Hildenbrandia were positioned on a well-supported branch associated with members of the Bangiales, indicating possible common ancestry. Structural analysis of the intron sequences revealed a signature structural feature in the P5b domain of intron 516 that is unique to all Bangialean introns in this position and not seen in intron 1506 or other group IC1 introns.
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PMID:A structural and phylogenetic analysis of the group IC1 introns in the order Bangiales (Rhodophyta). 1150 46

The second enzyme in the DNA base excision repair (BER) pathway, apurinic/apyrimidinic (AP) endonuclease or Ape1, hydrolyzes the phosphodiester backbone immediately 5' to an AP site generating a normal 3'-hydroxyl group and an abasic deoxyribose-5-phosphate, which is processed by subsequent enzymes of the BER pathway. AP sites are the most common form of DNA damage, and the persistence of AP sites in DNA results in a block to DNA replication, cytotoxic mutations, and genetic instability. Interestingly, Ape1/ref-1 is a multifunctional protein that not only is a DNA repair enzyme, but also functions as a redox factor maintaining transcription factors, such as Fos, Jun, nuclear factor-kappaB, PAX (paired box-containing family of genes), hypoxia inducible factor-lalpha (HIF-1alpha), HIF-1-like factor, and p53, in an active reduced state. Apel/ref-1 has also been implicated in a number of other activities, one of which is the activation of bioreductive drugs requiring reduction for activity. In this report, we present data supporting our findings that another level of posttranslational modification of Apel/ref-1 that clearly affects the AP endonuclease activity is the reduction or oxidation of this protein. Furthermore, we show data demonstrating that at least one of the sites involved in this redox regulation is the cysteine amino acid found at position 310, immediately adjacent to the crucial histidine residue at position 309 in the DNA repair active site. These findings suggest that the Apel/ref-1 protein may be much more intimately regulated at the posttranslational level than initially imagined.
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PMID:Redox regulation of the DNA repair function of the human AP endonuclease Ape1/ref-1. 1155 53

Endonucleases in DNA repair must be able to recognize damaged DNA as well as cleave the phosphodiester backbone. These functional prerequisites are manifested in very short patch repair (Vsr) endonuclease through a common endonuclease topology that has been tailored for recognition of TG mismatches. Structural and biochemical comparison with type II restriction enzymes illustrates how Vsr resembles these endonucleases in overall topology but also how Vsr diverges in terms of the detailed catalytic mechanism. A histidine and two metal-water clusters catalyze the phosphodiester cleavage. The mode of DNA damage recognition is also unique to Vsr. All other structurally characterized DNA damage-binding enzymes employ a nucleotide flipping mechanism for substrate recognition and for catalysis. Vsr, on the other hand, recognizes the TG mismatch as a wobble base pair and penetrates the DNA with three aromatic residues on one side of the mismatch. Thus, Vsr endonuclease provides important counterpoints in our understanding of endonucleolytic mechanisms and of damaged DNA recognition.
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PMID:The structural basis of damaged DNA recognition and endonucleolytic cleavage for very short patch repair endonuclease. 1155 9

Colicin endonucleases and the H-N-H family of homing enzymes share a common active site structural motif that has similarities to the active sites of a variety of other nucleases such as the non-specific endonuclease from Serratia and the sequence-specific His-Cys box homing enzyme I-PpoI. In contrast to these latter enzymes, however, it remains unclear how H-N-H enzymes cleave nucleic acid substrates. Here, we show that the H-N-H enzyme from colicin E9 (the E9 DNase) shares many of the same basic enzymological characteristics as sequence-specific H-N-H enzymes including a dependence for high concentrations of Mg2+ or Ca2+ with double-stranded substrates, a high pH optimum (pH 8-9) and inhibition by monovalent cations. We also show that this seemingly non-specific enzyme preferentially nicks double-stranded DNA at thymine bases producing 3'-hydroxy and 5'-phosphate termini, and that the enzyme does not cleave small substrates, such as dinucleotides or nucleotide analogues, which has implications for its mode of inhibition in bacteria by immunity proteins. The E9 DNase will also bind single-stranded DNA above a certain length and in a sequence-independent manner, with transition metals such as Ni2+ optimal for cleavage but Mg2+ a poor cofactor. Ironically, the H-N-H motif of the E9 DNase although resembling the zinc binding site of a metalloenzyme does not support zinc-mediated hydrolysis of any DNA substrate. Finally, we demonstrate that the E9 DNase also degrades RNA in the absence of metal ions. In the context of current structural information, our data show that the H-N-H motif is an adaptable catalytic centre able to hydrolyse nucleic acid by different mechanisms depending on the substrate and metal ion regime.
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PMID:Mechanism and cleavage specificity of the H-N-H endonuclease colicin E9. 1173 93

With the goal of developing artificial nucleases for DNA hydrolysis, metal-coordinating peptides have been tethered to a DNA-intercalating rhodium complex to deliver metal ions to the sugar-phosphate backbone. The intercalator, [Rh(phi)(2)bpy']Cl(3) [phi = 9,10-phenanthrenequinone diimine; bpy' = 4-(butyric acid)-4'-methyl-2,2'-bipyridine], provides DNA binding affinity, and a metal-binding peptide contributes reactivity. This strategy for DNA hydrolysis is a general one, and zinc(II)-promoted cleavage has been demonstrated for two widely different tethered metallopeptides. An intercalator coupled with a de novo-designed alpha helix containing two histidine residues has been demonstrated to cleave both supercoiled plasmid and linear DNA substrates. Mutation of this peptide confirms that the two histidine residues are essential for Zn(2+) binding and cleavage. Zinc(II)-promoted cleavage of supercoiled plasmid has also been demonstrated with an intercalator-peptide conjugate containing acidic residues and modeled after the active site of the BamHI endonuclease. Other redox-active metals, such as copper, have been delivered to DNA with our intercalator-peptide conjugates to effect oxidative chemistry. Copper cleavage experiments and photocleavage experiments with [Rh(phi)(2)bpy'](3+) complement the hydrolysis studies and provide structural information about the interactions between the tethered metallopeptides and DNA. Variation of the rhodium intercalator was also explored, but with a mismatch-specific intercalator, no site-specific hydrolysis was found. These experiments, in which the peptide, the metal cation, and the intercalator components of the conjugate are each varied, illustrate some of the issues involved in creating an artificial nuclease with DNA intercalators and metallopeptides.
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PMID:DNA hydrolysis and oxidative cleavage by metal-binding peptides tethered to rhodium intercalators. 1177 34

Long interspersed elements (LINEs) are ubiquitous genomic elements in higher eukaryotes. Here we develop a novel assay to analyze in vivo LINE retrotransposition using the telomeric repeat-specific elements SART1 and TRAS1. We demonstrate by PCR that silkworm SART1, which is expressed from a recombinant baculovirus, transposes in Sf9 cells into the chromosomal (TTAGG)n sequences, at the same specific nucleotide position as in the silkworm genome. Thus authentic retrotransposition by complete reverse transcription of the entire RNA transcription unit and occasional 5' truncation is observed. The retrotransposition requires conserved domains in both open reading frames (ORFs), including the ORF1 cysteine- histidine motifs. In contrast to human L1, recognition of the 3' untranslated region sequence is crucial for SART1 retrotransposition, which results in efficient trans-complementation. Swapping the endonuclease domain from TRAS1 into SART1 converts insertion specificity to that of TRAS1. Thus the primary determinant of in vivo target selection is the endonuclease domain, suggesting that modified LINEs could be used as gene therapy vectors, which deliver only genes of interest but not retrotransposons themselves in trans to specific genomic locations.
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PMID:Transplantation of target site specificity by swapping the endonuclease domains of two LINEs. 1182 33


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