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:3.1.25.1 (deoxyribonuclease)
1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

T4N5 liposomes, which contain the DNA repair enzyme T4 endonuclease V, were applied to mouse skin in vivo and added to cultured murine keratinocytes in vitro. The fate of the liposome membrane was followed using a fluorescent, lipophilic dye, and the fate of the enzyme was traced by immunogold labeling, followed by brightfield, fluorescence, or transmission electron microscopy. In vivo, T4N5 liposomes penetrated the stratum corneum, localized in epidermis and appendages of the skin, and were found inside basal keratinocytes. The enzyme was found inside keratinocytes treated in vitro and in the epidermis, hair follicles, and sebaceous glands of topically treated skin. Ultrastructural studies demonstrated the presence of liposomes in the cytoplasm of cells in the epidermis often concentrated in a perinuclear location. The enzyme was present in both nucleus and cytoplasm of keratinocytes and Langerhans cells. Liposomes were found in cells of the lymph nodes draining the site of contact sensitization, in association with topically applied antigen. The results demonstrate that liposomes can deliver encapsulated proteins into cells of the skin in vivo and provide insight into how liposome-enhanced DNA repair reduces UV-induced skin cancer and systemic immunosuppression in mice.
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PMID:Localization of liposomes containing a DNA repair enzyme in murine skin. 793 Jun 68

Tanning is a protective response of ultraviolet (UV)-irradiated skin that decreases damage from subsequent sun exposures by increasing the epidermal content of melanin, a brown-black pigment that absorbs light energy throughout the UV and visible portions of the electromagnetic spectrum. The melanin pigment is made by epidermal melanocytes and transferred to surrounding keratinocytes. The action spectrum, time course, and histologic features of tanning are well studied, but the initiating molecular events are unknown. Previous work has shown that T4 endonuclease V, a prokaryotic DNA repair enzyme that catalyzes the first and rate-limiting step in repair of UV-induced pyrimidine dimers, delivered in carrier liposomes (T4N5), enhances repair of UV-induced DNA damage in cultured human cells and protects against photocarcinogenesis in an animal model. We now report that T4N5 treatment enhances UV-induced melanogenesis, as measured by melanin content, tyrosinase activity, 14C-dopa incorporation, and visual assessment in both S91 murine melanoma cells and human melanocytes. T4N5 treatment also increases cell yields following UV irradiation. These data suggest that tanning can be stimulated through enhanced DNA repair.
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PMID:Treatment of human melanocytes and S91 melanoma cells with the DNA repair enzyme T4 endonuclease V enhances melanogenesis after ultraviolet irradiation. 822 26

Reductive methylation of the alpha NH2 moiety of the DNA repair enzyme T4 endonuclease V has been shown previously to eradicate both the N-glycosylase and apyrimidinic/apurinic lyase activities of the enzyme (Schrock, R. D., III, and Lloyd, R. S. (1991) J. Biol. Chem. 266, 17631-17639). The present study uses the technique of site-directed mutagenesis to investigate the important parameters involved in the cleavage mechanism. The prediction was that the addition of an amino acid in the immediate NH2-terminal region of the protein would alter the proximity of the alpha NH2 moiety of Thr2 to its target, thereby severely compromising the enzyme's catalytic activity. However, substitutions in this region generally should be tolerated. To test this hypothesis, three substitutions of the NH2-terminal amino acid were produced: Ser2 (T2S), Val2 (T2V), and Pro2 (T2P). An addition mutant was also produced by adding a glycine between the first and second amino acids of the protein (Thr2-Gly-Arg3) (+Gly). The T2P and +Gly mutants had negligible pyrimidine dimer-specific N-glycosylase activity as well as negligible pyrimidine dimer-specific nicking activity in vitro. Conversely, the T2S enzyme exhibited wild type levels of activity and the T2V exhibited intermediate levels of activity in vitro. Results from ultraviolet (UV) survival studies of the mutant enzymes indicated that the in vivo activities of these enzymes were directly correlated to the enzymes' ability to cleave at pyrimidine dimers in vitro. These results indicate that a critical parameter for the functionality of endonuclease V is the relative distance between the primary alpha NH2 group in the active site of the enzyme and those elements responsible for DNA binding and pyrimidine dimer recognition.
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PMID:Site-directed mutagenesis of the NH2 terminus of T4 endonuclease V. The position of the alpha NH2 moiety affects catalytic activity. 841 66

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 A resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60 degree inclination at the central thymine dimer. The adenine base complementary to the 5' side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.
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PMID:Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition. 852 94

Any uracil bases in DNA, a result of either misincorporation or deamination of cytosine, are removed by uracil-DNA glycosylase (UDG), one of the most efficient and specific of the base-excision DNA-repair enzymes. Crystal structures of human and viral UDGs complexed with free uracil have indicated that the enzyme binds an extrahelical uracil. Such binding of undamaged extrahelical bases has been seen in the structures of two bacterial methyltransferases and bacteriophage T4 endonuclease V. Here we characterize the DNA binding and kinetics of several engineered human UDG mutants and present the crystal structure of one of these, which to our knowledge represents the first structure of any eukaryotic DNA repair enzyme in complex with its damaged, target DNA. Electrostatic orientation along the UDG active site, insertion of an amino acid (residue 272) into the DNA through the minor groove, and compression of the DNA backbone flanking the uracil all result in the flipping-out of the damaged base from the DNA major groove, allowing specific recognition of its phosphate, deoxyribose and uracil moieties. Our structure thus provides a view of a productive complex specific for cleavage of uracil from DNA and also reveals the basis for the enzyme-assisted nucleotide flipping by this critical DNA-repair enzyme.
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PMID:A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. 890 Feb 70

Ultraviolet B (UVB) component of the sunlight is the major cause of nonmelanoma skin cancer (NMSC) in humans. UVB is absorbed directly by cellular DNA and produces lesions that may cause mutation(s) in target gene(s) ultimately leading to cancer. Early detection of these lesions, therefore, may help to identify individuals at a high risk to develop NMSC, and devise approaches for the prevention of this common malignancy. Employing mouse skin as a model, we applied a 32P postlabelling method to detect UVB-induced DNA lesions in the epidermis in nanomole quantities. Autoradiography maps showed that epidermal DNA from UVB exposed mice at 24 h contain up to five DNA lesions; the quantitation of these lesions showed that their formation increased in a UVB dose-dependent manner. Treatment of DNA samples with the bacteriophage DNA repair enzyme T4 endonuclease V confirmed that four of these lesions are pyrimidine dimers. While, some of these lesions were repaired 18 h after UVB irradiation, 30% of them persisted even 48 h post-irradiation. Application of a sunscreen containing ethylhexyl-p-methoxycinnamate or chemopreventive agent green tea polyphenols or silymarin to the skin of the mice prior to UVB exposure was found to prevent the formation of pyrimidine dimers.
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PMID:Ultraviolet B radiation-induced DNA lesions in mouse epidermis: an assessment using a novel 32P-postlabelling technique. 895 42

The process of moving a DNA base extrahelical (base flipping) has been shown in the co-crystal structure of a UV-induced pyrimidine dimer-specific glycosylase, T4 endonuclease V, with its substrate DNA. Compared with other enzymes known to use base flipping, endonuclease V is unique in that it moves the base opposite the target site extrahelical, rather than moving the target base itself. Utilizing substrate analogs and catalytically inactive mutants of T4 endonuclease V, this study investigates the discrete steps involved in damage recognition by this DNA repair enzyme. Specifically, fluorescence spectroscopy analysis shows that fluorescence changes attributable to base flipping are specific for only the base directly opposite either abasic site analogs or the 5'-thymine of a pyrimidine dimer, and no changes are detected if the 2-aminopurine is moved opposite the 3'-thymine of the pyrimidine dimer. Interestingly, base flipping is not detectable with every specific binding event suggesting that damage recognition can be achieved without base flipping. Thus, base flipping does not add to the stability of the specific enzyme-DNA complex but rather induces a conformational change to facilitate catalysis at the appropriate target site. When used in conjunction with structural information, these types of analyses can yield detailed mechanistic models and critical amino acid residues for extrahelical base movement as a mode of damage recognition.
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PMID:The role of base flipping in damage recognition and catalysis by T4 endonuclease V. 934 Nov 65

Ultraviolet light induces the expression of tumor necrosis factor alpha (TNF alpha) in many mammalian cells. We have examined the signal for this induction in a human DNA repair-deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV-irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V increased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane-bound TNF alpha expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV-irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNF alpha gene expression in mouse and human cells.
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PMID:UV-DNA damage in mouse and human cells induces the expression of tumor necrosis factor alpha. 964 8

A new approach to photoprotection is to repair DNA damage after UV exposure. This can be accomplished by delivery of a DNA repair enzyme with specificity to UV-induced cyclobutane pyrimidine dimers into skin by means of specially engineered liposomes. Treatment of DNA-repair-deficient xeroderma pigmentosum patients or skin cancer patients with T4N5 liposome lotion containing such DNA repair liposomes increases the removal of DNA damage in the first few hours after treatment. In these studies, a DNA repair effect was observed in some patients treated with heat-inactivated enzyme. Unexpectedly, it was discovered that the heat-inactivated T4 endonuclease V enzyme refolds and recovers enzymatic activity. These studies demonstrate that measurements of molecular changes induced by biological drugs are useful adjuvants to clinical studies.
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PMID:Photoprotection by topical DNA repair enzymes: molecular correlates of clinical studies. 1004 8

Exposing human skin to ultraviolet radiation causes DNA damage, sunburn, immune alterations, and eventually, skin cancer. We wished to determine whether liposomes containing a DNA repair enzyme could prevent any of the acute effects of irradiation when applied after ultraviolet exposure. Fifteen human patients with a prior history of skin cancer were exposed to two minimal erythema doses of ultraviolet radiation on their buttock skin. Liposomes containing T4 endonuclease V or heat-inactivated enzyme were applied immediately and at 2, 4, and 5 h after ultraviolet irradiation. Transmission electron microscopy after anti-T4 endonuclease V-staining and immunogold labeling on biopsies taken at 6 h after ultraviolet exposure revealed that the enzyme was present within cells in the skin. Immunohistochemical DNA damage studies suggested a trend toward improved DNA repair at the active T4 endonuclease V liposome-treated test sites. Although the active T4 endonuclease V liposomes did not significantly affect the ultraviolet-induced erythema response and microscopic sunburn cell formation, they nearly completely prevented ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha RNA message and of interleukin-10 protein. These studies demonstrate that liposomes can be used for topical intracellular delivery of small proteins to human skin and suggest that liposomes containing DNA repair enzymes may provide a new avenue for photoprotection against some forms of ultraviolet-induced skin damage.
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PMID:Topical treatment with liposomes containing T4 endonuclease V protects human skin in vivo from ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha. 1062 Jan 31


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