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)

Apurinic/apyrimidinic (AP) sites are mutagenic and block DNA synthesis in vitro. Repair of AP sites is initiated by AP endonucleases that cleave just 5' to the damage. We linked a 4.1-kilobase pair HindIII DNA fragment from the region upstream of the human AP endonuclease gene (APE) to the chloramphenicol acetyltransferase (CAT) gene. Deletions generated constructs containing 1.9 kilobase pairs to 50 base pairs (bp) of the APE upstream region. Transient transfection studies in HeLa cells established that the basal APE promoter is contained within a 500-bp fragment. The major transcriptional start site in HeLa, hepatoma (HepG2), and myeloid leukemic (K562) cells was mapped to a cluster of sites approximately 130 bp downstream of a putative "CCAAT box," approximately 130 bp 5' of the first splice junction in APE. Deletion of 5' sequences to within 10 bp of the CCAAT box reduced the CAT activity by only about half, and removal of the CCAAT box region left a residual promotor activity approximately 9%. Deletion to 31 bp upstream of the transcriptional start site abolished APE promoter activity. DNA sequence analysis revealed potential transcription factor recognition sites in the APE promoter. Gel mobility-shift assays showed that both human upstream factor and Sp1 can bind their respective sites in the APE promoter. However, DNase I footprinting using HeLa nuclear extract showed that the binding of Sp1 and upstream factor is blocked by the binding of other proteins to the nearby CCAAT box region.
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PMID:Characterization of the promoter region of the human apurinic endonuclease gene (APE). 753 97

The preferred cleavage sites in dsDNA and ssDNA for the extracellular Serratia marcescens endonuclease (commercially available as BENZONASE) were identified by limited digestion of PCR-generated substrates. Two different dsDNA substrates were synthesized by using either radioactively or fluorescent dye labeled primers. ssDNA of identical sequence to one of the fluorescent dye labeled duplex strands was prepared by affinity chromatography. Cleavage experiments carried out under single hit conditions demonstrate that the enzyme shows preferences for GC-rich regions in dsDNA, in particular d(G).d(C)-tracts, and avoids cleavage of d(A).d(T)-tracts. There is a correlation between cleavage at a given position in one strand with cleavage at the same position in the other strand of the duplex. ssDNA cleavage occurs at somewhat different preferred sites than observed in dsDNA. On dsDNA, the Serratia nuclease produces a very different cleavage pattern compared to bovine pancreatic DNase I, with the notable exception that both enzymes avoid d(A).d(T)-tracts. In general, the Serratia nuclease compared to DNase I is a slightly more nonspecific endonuclease that attacks a particular substrate more evenly under standard reaction conditions. At high ionic strength or in the presence of DMSO, it becomes more nonspecific. Addition of urea, however, makes the enzyme more selective than observed under standard conditions. From these results which were confirmed by the results of cleavage experiments with synthetic oligodeoxynucleotides, we conclude that the Serratia nuclease like DNase I is sensitive to global features of the DNA, for example, the width of the minor groove. In addition, localized sequence-dependent interactions between substrate and nuclease determine whether a site is cleaved preferentially. Some of these interactions seem to be the same for ds- and ssDNA.
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PMID:Sequence preferences in cleavage of dsDNA and ssDNA by the extracellular Serratia marcescens endonuclease. 754 35

Activation of a triplet of nuclear proteins (NP42-50) was observed in human Jurkat T cell line following treatment with an antibody to CD95 (Fas/Apo-1), a cell surface molecule involved in apoptotic cell death. The nuclease activity, corresponding to a triplet of proteins observed at approximately 42, 45, and 50 kDa in size, was extractable, heat-stable, and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing deoxyribonucleic acids (SDS-PAGE-DNA) assay. The NP42-50 activity requires the presence of Mg2+/Ca2+ and is insensitive to inactivation by heating at 80 degrees C for 5 min. Zinc effectively inhibited the enzymatic activity of NP42-50 on SDS-PAGE-DNA and also protected Jurkat cells from the CD95-mediated apoptosis in cell cultures. The nuclease activation, however, was not a unique pathway for the CD95-mediated cell death. The apoptosis induced by arabinofuranosyl cytosine, a chemotherapeutic agent, also activated the NP42-50 nuclease activity in Jurkat cells, suggesting that a similar cascade of subsequent events in apoptosis may occur in most instances although many different signals can initiate apoptotic cell death in various cell types. The nuclease identified by this study appears to be distinguishable from DNase I or DNase II by its molecular characteristics and its enzymatic requirements. The NP42-50, with respect to the nuclease activity closely associated with apoptotic cell death, may serve as a candidate for the endonuclease(s) involved in the cleavage of DNA into fragments during apoptosis.
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PMID:A triplet of nuclease proteins (NP42-50) is activated in human Jurkat cells undergoing apoptosis. 755 79

The binding of the MboII restriction endonuclease (R.MboII; ENase) to DNA containing its recognition site was investigated using a mobility shift assay. R.MboII forms specific, stable and immunodetectable complexes with its canonical target sequence. The association constant (Ka) of R.MboII was calculated to be 2.8 x 10(9)/M, and is about 10(4)-fold higher than the Ka value for non-specific binding. Based on results obtained after sedimentation of the R.MboII-DNA complex in a glycerol gradient and measurement of the retardation of the complexes in polyacrylamide gels, we conclude that specific binding to the canonical sequence involves a monomer of R.MboII. DNase I footprinting has shown that the enzyme covers 16 nucleotides of DNA on the 5'-GAAGA-3' strand.
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PMID:Interaction of the MboII restriction endonuclease with DNA. 760 88

The objectives of this study were to elucidate the structural organization of the gene for human deoxyribonuclease I (DNase I) and to identify the mutation site underlying its classical genetic polymorphism. In order to determine the organization of this gene, we utilized a combination of direct polymerase chain reaction (PCR)-amplification of human genomic DNA and isolation of the overlapping clones from a cosmid human genomic library. Restriction endonuclease mapping, Southern blotting and DNA sequencing showed that the DNase I gene was approximately 3.2 kilobases long, it comprised 9 (I-IX) exons separated by eight introns and its complete sequence was determined. The first exon contained only the non-translated sequences of mRNA. In addition to several putative regulatory elements, TATA-like and CAAT-like sequences were observed in the region upstream of the translation initiation codon. These results provide information that will help to understand the expression and regulation of DNase I. The isoelectric focusing patterns of human DNase I showed that it exhibits classical genetic polymorphism (Kishi et al. 1989, 1990). A comparison of the entire translated sequences of the DNase I gene from two pairs of individuals with common DNase I phenotypes 1 and 2 revealed only one nucleotide residue difference in exon VIII, A for phenotype 1 and G for phenotype 2, thus producing Gln and Arg amino acid substitutions respectively at position 222 from the NH2-terminus of the mature enzyme. The predicted charge changes attributable to these amino acid substitutions are entirely consistent with the isoelectric focusing profiles of these two DNase I isozymes. We conclude that this substitution is solely responsible for the classical polymorphism of DNase I protein.
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PMID:Structure of the human deoxyribonuclease I (DNase I) gene: identification of the nucleotide substitution that generates its classical genetic polymorphism. 776 78

The beta-thymosins are a family of < 5kDa (MW), mostly acidic, proteins which were originally defined in the immune system. Recently, specific members of this family of cytoplasmic polypeptides, namely beta-4 and beta-10, were shown to bind monomeric G-actin both in vitro and in vivo. Whilst many aspects of programmed cell death or 'apoptosis' remain to be defined, the Ca2+/Mg(2+)-dependent endonuclease, DNase I does feature in this process. Monomeric G-actin binds to and inhibits the DNA-degrading activity of DNase I. Given that the intracellular abundance of thymosins beta-4 and beta-10 is related to cell division and differentiation and that anticancer/morphogenic agents such as retinoic acid (RA) and cyclic AMP modulate expression of their respective genes, it is possible that these G-actin sequestering proteins play significant roles in apoptosis perhaps mediated via DNase I.
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PMID:Molecular interactions between G-actin, DNase I and the beta-thymosins in apoptosis: a hypothesis. 781 61

A highly effective method consisting of two affinity chromatography steps and ion-exchange and gel-filtration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg(2+)-dependent single-strand multiple nicking of the substrate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (Kobsm = 43 nM) and considerable catalytic efficiency (kappcat/Kobsm = 0.32 min-1.nM-1) of the reaction.
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PMID:Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique. 781 27

Using transgenic mice, we have defined novel gene regulatory elements, termed "facilitators." These elements bilaterally flank, by up to 1 kb, a 200-bp T-cell-specific enhancer domain in the human adenosine deaminase (ADA) gene. Facilitators were essential for gene copy-proportional and integration site-independent reporter expression in transgenic thymocytes, but they had no effect on the enhancer in transfected T cells. Both segments were required. Individual segments had no activity. A lack of facilitator function caused positional susceptibility and prevented DNase I-hypersensitive site formation at the enhancer. The segments were required to be at opposed ends of the enhancer, and they could not be grouped together. Reversing the orientation of a facilitator segment caused a partial loss of function, suggesting involvement of a stereospecific chromatin structure. trans-acting factor access to enhancer elements was modeled by exposing nuclei to a restriction endonuclease. The enhancer domain was accessible to the 4-cutter DpnII in a tissue- and cell-type-specific fashion. However, unlike DNase I hypersensitivity and gene expression, accessibility to the endonuclease could occur without the facilitator segments, suggesting that an accessible chromatin domain is an intermediate state in the activational pathway. These results suggest that facilitators (i) are distinct from yet positionally constrained to the enhancer, (ii) participate in a chromatin structure transition that is necessary for the DNase I hypersensitivity and the transcriptional activating function of the enhancer, and (iii) act after cell-type-specific accessibility to the enhancer sequences is established by factors that do not require the facilitators to be present.
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PMID:Dissecting a locus control region: facilitation of enhancer function by extended enhancer-flanking sequences. 782 28

UV-induced cyclobutane dimers and 6-4 photoproducts, containing an unmodified nucleotide at the 5'-position were released from DNA by means of digestion with DNase I, snake venom phosphodiesterase and prostatic acid phosphatase. The enzymes were deactivated by proteinase K followed by ethanol precipitation. The products were phosphorylated by polynucleotide kinase and [gamma-32P]ATP. The TLC system used for the analysis enables separation of the different photoproducts and detection at a fmol level. T4 endonuclease treatment was applied to confirm the positions of cyclobutane dimers.
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PMID:Analysis of UV-induced DNA photoproducts by 32P-postlabelling. 783 95

Replication of the palindromic inverted terminal repeats (ITRs) of adeno-associated virus type 2 requires several functions of the viral nonstructural Rep proteins. These include binding to the ITR, nicking of the double-stranded replication intermediate at the terminal resolution site (trs), and then strand displacement and synthesis from the nick. This report demonstrates the ability of both recombinant fusion maltose-binding protein (MBP)-Rep68 delta produced in Escherichia coli and wild-type (wt) Rep68 to bind to a linear truncated form of the ITR, delta 57 ITR, with similar affinity as to the wt hairpin ITR. A dissociation constant for MBP-Rep68 delta of approximately 8 x 10(-10) M was determined for the wt ITR and delta 57 ITR probes. Truncation of delta 57 ITR to generate delta 28 ITR, which retains the GCTC repeat motif but not the trs, bound at least 10 times less efficiently than delta 57 ITR. Extension of delta 28 ITR with nonspecific sequence restored the ability of MBP-Rep68 delta to bind to delta 28 ITR. Thus, high-affinity binding would appear to require stabilization by flanking sequence as well as the intact GCTC repeat motif. Cleavage of the delta 57 ITR probe with DdeI, which truncates the flanking sequence and was previously shown to inhibit binding by Rep68, also inhibited the binding of MBP-Rep68 delta. The requirements for stable binding were further defined with a series of oligonucleotide probes which spanned the region protected by MBP-Rep78 in DNase I footprinting. The binding activity of either MBP-Rep68 delta or wt Rep68 to hairpin ITR or delta 57 ITR was indistinguishable. However, the binding activity of MBP-Rep68 delta to DNA does not appear to correlate with trs endonuclease activity. The nicking and covalent linkage of MBP-Rep68 delta to the nonhairpin delta 57 ITR was approximately 100-fold less efficient than its linkage to a hairpin-containing ITR. Therefore, although the hairpin portion of the ITR does not appear to play a role in recognition and stabilization of MBP-Rep68 delta binding, its presence does affect the trs cleavage activity of the protein.
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PMID:Sequence requirements for stable binding and function of Rep68 on the adeno-associated virus type 2 inverted terminal repeats. 793 28


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