Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.21.3 (deoxyribonuclease)
 1,528 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly(ethylenimine) (PEI) is a synthetic polycation that has been used successfully for gene delivery both in vitro and in vivo due, in theory, to a form of protection that is afforded to the carried plasmids. In this study the stability of PEI/DNA complexes was demonstrated using deoxyribonuclease (DNase) 1 and DNase 2, various levels of pH, and increasing exposure times. DNA that was complexed with PEI was not degraded when exposed to at least 25 Units of either enzyme for 24 h while uncomplexed forms of the same plasmid were digested when exposed to 0.010 Units of DNase 1 for 0.05 h or 0. 003 Units of DNase 2 for 1 h. For further comparison, the stability of complexes made with poly(L-lysine) (PLL) and DNA was examined and found to be lower than that of PEI/DNA complexes; PLL-complexed DNA was digested on exposure to 1.25 Units of DNase 1 for 3 min. Cells were transfected with PEI/DNA complexes and, by using a pH indicator and optical recording techniques, it was found that the normal lysosomal pH value of 5.0 was not altered, bringing into question PEI's hypothesized lysosomal entry. Confocal microscopy showed that PEI/DNA complexes and lysosomes do not merge during transfection (although PLL/DNA complexes do). The lack of lysosomal involvement in PEI-mediated transfection is surprising because it goes against the conventional wisdom that has attempted to explain how PEI functions during transfection. PEI forms a stable complex with DNA, which moves from endocytosis to nuclear entry without significant cellular obstacles.
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PMID:Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery. 1088 73

Nuclear targeting of bacterial proteins is an emerging pathogenic mechanism whereby bacterial proteins can interact with nuclear molecules and alter the physiology of host cells. The fully sequenced bacterial genome can predict proteins that target the nuclei of host cells based on the presence of nuclear localization signal (NLS). In the present study, we predicted bacterial proteins with the NLS sequences from Klebsiella pneumoniae by bioinformatic analysis, and 13 proteins were identified as carrying putative NLS sequences. Among them, HsdM, a subunit of KpnAl that is a type I restriction-modification system found in K. pneumoniae, was selected for the experimental proof of nuclear targeting in host cells. HsdM carried the NLS sequences, (7)KKAKAKK(13), in the N-terminus. A transient expression of HsdM-EGFP in COS-1 cells exhibited exclusively a nuclear localization of the fusion proteins, whereas the fusion proteins of HsdM with substitutions in residues lysine to alanine in the NLS sequences, (7)AAAKAAA(13), were localized in the cytoplasm. HsdM was co-localized with importin o in the nuclei of host cells. Recombinant HsdM alone methylated the eukaryotic DNA in vitro assay. Although HsdM tested in this study has not been considered to be a virulence factor, the prediction of NLS motifs from the full sequenced genome of bacteria extends our knowledge of functional genomics to understand subcellular targeting of bacterial proteins.
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PMID:Prediction of bacterial proteins carrying a nuclear localization signal and nuclear targeting of HsdM from Klebsiella pneumoniae. 1985 38

Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains. In the present work, molecular dynamics simulations are used to explore this proposal. Molecular dynamics simulations suggest that the Lys-ATP contact alternates with a contact with a nearby loop housing the conserved QxxxY motif that had been implicated in DNA cleavage. This model is tested here using in vivo and in vitro experiments. The results indicate how local interactions are transduced to domain motions within the endonuclease/motor subunit.
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PMID:Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I. 2497 99

DNA phosphorothioate (PT) modification is a recently identified epigenetic modification that occurs in the sugar-phosphate backbone of prokaryotic DNA. Previous studies have demonstrated that DNA PT modification is governed by the five DndABCDE proteins in a sequence-selective and RP stereo-specific manner. Bacteria may have acquired this physiological modification along with dndFGH as a restriction-modification system. However, little is known about the biological function of Dnd proteins, especially the smallest protein, DndE, in the PT modification pathway. DndE was reported to be a DNA-binding protein with a preference for nicked dsDNA in vitro; the binding of DndE to DNA occurs via six positively charged lysine residues on its surface. The substitution of these key lysine residues significantly decreased the DNA binding affinities of DndE proteins to undetectable levels. In this study, we conducted site-directed mutagenesis of dndE on a plasmid and measured DNA PT modifications under physiological conditions by mass spectrometry. We observed distinctive differences from the in vitro binding assays. Several mutants with lysine residues mutated to alanine decreased the total frequency of PT modifications, but none of the mutants completely eliminated PT modification. Our results suggest that the nicked dsDNA-binding capacity of DndE may not be crucial for PT modification and/or that DndE may have other biological functions in addition to binding to dsDNA.
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PMID:In vivo mutational characterization of DndE involved in DNA phosphorothioate modification. 2526 84


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