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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ruvA and ruvB genes of Escherichia coli encode a novel DNA helicase that interacts with Holliday junctions and promotes branch migration. In this work, we have investigated the protein-DNA complexes formed between RuvA, RuvB and Holliday junctions. As shown previously, RuvA protein binds a synthetic Holliday junction in vitro, to form a specific protein-DNA complex that can be detected by a band-shift assay. We now show that the combined presence of RuvA and RuvB results in a super-shift of this complex indicative of the formation of a RuvAB-Holliday junction complex. In the absence of RuvA, the RuvB protein fails to bind Holliday junctions. The RuvAB-Holliday junction complex was detected by the band-shift assay only under conditions that favoured its stability, e.g. complex formation in the presence of a nucleoside triphosphate that can not be hydrolysed by RuvB (adenosine 5'-[gamma-thio]triphosphate). In contrast, nucleoside triphosphates that can be hydrolysed (ATP, dATP, dCTP or TTP), lead to RuvAB-mediated branch migration of the junction. These results indicate that the formation of a (RuvAB-ATP)-Holliday junction complex represents the first step in the process of branch migration, and that branch migration is dependent upon ATP hydrolysis. In addition, we show that Holliday junction DNA stimulates the ATPase activity of RuvAB to a greater extent than either single-stranded or linear duplex DNA.
J Mol Biol 1993 Jul 20
PMID:Formation of a RuvAB-Holliday junction complex in vitro. 839 34

To characterize the process by which the mammalian nucleotide excision repair complex interacts with DNA to recognize and repair lesions, we have investigated the size and distribution of repair patches induced by human cell extracts in ultraviolet light-irradiated plasmid DNA. Repair synthesis was carried out in a buffer substituting biotinylated dUTP for dTTP, to allow repair patches to be detected by electron microscopy after streptavidin/colloidal gold labelling. Individual repair events on circular plasmids that had undergone repair synthesis in cell extracts were scored as gold particles bound specifically to irradiated molecules. Samples of over 2000 irradiated and unirradiated plasmids were counted. Repair synthesis at ultraviolet light photoproducts typically replaced about 30 nucleotides, since 69% of patches contained only one particle of 10 nm gold and 24% of patches contained two gold particles (each covering approx. 29 nucleotides). In addition, the ordering of repair events among damaged plasmids closely fitted a Poisson distribution, indicating that repair of lesions is achieved via a non-processive, random diffusion mechanism. This suggests that the repair complex is not intrinsically processive.
J Mol Biol 1993 May 20
PMID:Electron microscopy of DNA excision repair patches produced by human cell extracts. 851 Jan 46

2'-Deoxythymidine 5'-triphosphate and 2'-deoxyadenosine 5'-triphosphate analogs containing a methylene group between the alpha phosphorus and 5' oxygen were synthesized. The substrate properties of these compounds toward some mammalian DNA polymerases and retroviral reverse transcriptases were evaluated using a system containing phage M13mp10 DNA, a synthetic oligonucleotide, and the enzyme. The compounds containing a hydroxyl at the 3' position were incorporated into the DNA chain by DNA polymerase alpha and terminal deoxynucleotidyl transferase, but were not recognized by retroviral reverse transcriptases and mammalian DNA polymerases epsilon and beta. The selectivity of the compounds synthesized was capitalized on during simultaneous isolation of DNA polymerases alpha and epsilon from human placenta. A methylene group was also introduced into the acyclovir molecule. It was shown that this modification inactivates furanose-related nucleotide analogs, but has a minor effect on the substrate properties of acyclic nucleotide analogs.
Mol Biol (Mosk)
PMID:[New nucleotide inhibitors of human DNA polymerase alpha]. 855 70

We describe a new method for random mutagenesis of DNA based on the use of a mixture of triphosphates of nucleoside analogues. The method relies on DNA amplification in vitro with Taq polymerase and in the presence of the 5'-triphosphates of 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C] [1,2]oxazin-7-one(dP) and of 8-oxo-2' deoxyguanosine (8-oxodG). The newly synthesised triphosphate derivative of dP (dPTP) is an excellent substrate for Taq polymerase (Km = 22 microM versus Km = 9.5 microM for TTP); it is incorporated in place of TTP and, with a approximately fourfold lower efficiency, in place of dCTP. After 30 cycles of DNA amplification, equimolar mixtures of the four normal dNTPs and dPTP yield the following frequencies of the four transition mutations: A-->G (4.4 x 10(-2), T-->C (4.3 x 10(-2), G-->A (1.1 x 10(-2) and C-->T (1.0 x 10(-2). The triphosphate derivative of 8-oxodG (8-oxodGTP) is incorporated opposite template adenine and yields two transition mutations (A-->C and T-->G) at frequencies of 0.8 x 10(-2) and 1.2 x 10(-2) respectively. Reaction mixtures containing dPTP and 8-oxodGTP results in both dP and 8-oxodG-induced mutations and an extensive array of codon changes in the absence of insertions and deletions. The method described differs from previous mutagenesis procedures in three respects: (1) it enables very high frequencies of base substitutions (up to 1.9 x 10(-1) (2) it allows control of the mutational load via the number of DNA amplification cycles and (3) it yields both transition and transversion mutations. The procedure may find application in the generation of libraries of DNA and protein mutants from which species with improved or novel activities may be selected.
J Mol Biol 1996 Feb 02
PMID:An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. 856 99

The anti-human immunodeficiency virus (anti-HIV) agent 2',3'-didehydro-3'-deoxythymidine (D4T), like other 2',3'-dideoxynucleosides, requires conversion to its 5'-triphosphate to exert its pharmacological effect. Although D4T-triphosphate is unusually potent as an inhibitor of HIV-1 reverse transcriptase, the phosphorylation of the drug at low dose levels is inefficient because of its low affinity as an alternate substrate for the initial phosphorylation enzyme thymidine kinase. Because thymidine kinase is under feedback regulatory control by the physiological deoxynucleoside-5'-triphosphate dTTP, we examined the effect on D4T phosphorylation and thus, potentially, on its antiviral activity, of a variety of agents that lower intracellular dTTP pools. We found that agents that inhibit the de novo pyrimidine biosynthetic pathway have the ability to increase D4T phosphorylation, the most effective being two inhibitors of thymidylate formation, methotrexate and 5-fluoro-2'-deoxyuridine, compounds that block the enzymes dihydrofolate reductase and thymidylate synthetase, respectively. Because HIV itself lacks the capacity to synthesize dTTP and the other deoxynucleoside triphosphates essential for viral replication, combinations of D4T with modulatory agents that deplete host-cell dTTP, unlike conventional anti-HIV drug monotherapy directed solely at viral enzymes, have the ability to inhibit replication of mutant HIV strains as well as of wild-type virus.
Mol Pharmacol 1996 Jul
PMID:2',3'-Didehydro-3'-deoxythymidine: regulation of its metabolic activation by modulators of thymidine-5'-triphosphate biosynthesis. 870 Jan 8

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying DNA that is based upon the combined action of a DNA polymerase and restriction enzyme. Previously, a form of SDA was developed which utilizes the exonuclease deficient Klenow fragment of E. coli polymerase I (exo Klenow) and the restriction enzyme HincII to achieve 10(8)-fold amplification in 2 h at 37 degrees C (Walker, G.T., 1993, PCR Methods and Applications 3; 1-6). A new thermophilic form of SDA is reported here which uses a restriction endonuclease from Bacillus stearothermophilus (BsoBI) and a 5'-->3' exonuclease deficient polymerase from Bacillus caldotenax (exo Bca). SDA was used to amplify DNA from Mycobacterium tuberculosis. An amplification factor of 10(10)-fold was achieved after 15 min of SDA at 60 degrees C. The new thermophilic system is much more specific than the previous mesophilic system as evidenced by a dramatic decrease in background amplification products. Thermophilic SDA was also optimized with dUTP substituted for TTP to enable amplicon decontamination using uracil-DNA glycosylase.
Mol Cell Probes 1996 Aug
PMID:Detection of M. tuberculosis DNA using thermophilic strand displacement amplification. 886 73

A 44-year-old women was treated for hyperparathyroidism resulting from parathyroid hyperplasia. Several months later, following a flu-like episode, she developed fever, confusion, abdominal pain, and diffuse petechiae, with severe thrombocytopenia and hemolytic anemia. She died on the 11th day of hospitalization. At autopsy she had multiple endocrine neoplasia type I, with two islet cell tumors, adrenal adenoma, pituitary adenoma, and bronchial carcinoid with liver metastasis. Florid visceral microthrombi involved arterioles and capillaries of the heart, including the conduction system. Brain, kidney, pancreas, adrenal, and portal areas of the liver were also heavily involved, but thrombi were rare in the liver sinusoids and the lungs. PAS-positive subendothelial deposits were demonstrated. In spite of the disseminated malignancy, the morphologic and laboratory findings were inconsistent with disseminated intravascular coagulation (DIC), and supported the clinical diagnosis of TTP. To the best of our knowledge this is the first report association of TTP with MEN and raises the question of a genetic linkage and/or hormonal interaction.
Hematopathol Mol Hematol 1996
PMID:Fatal thrombotic thrombocytopenic purpura (TTP) presenting concurrently with metastatic multiple endocrine neoplasia (MEN) type I. 887 34

We demonstrate that human 2'-deoxycytidine kinase (dCK) is a nonenantioselective enzyme because it phosphorylates beta-D-2'-deoxycytidine (D-dCyd), the natural substrate, and beta-L-2'-deoxycytidine (L-dCyd), its enantiomer, with the same efficiency. Kinetic studies showed that L-dCyd is a competitive inhibitor of the phosphorylation of D-dCyd with a Kl value of 0.12 microM, which is lower than the K(m) value for D-dCyd (1,2 microM). Chemical modifications of either the base or the pentose ring strongly decrease the inhibitory potency of L-dCyd, L-dCyd is resistant to cytidine deaminase and competes in cell cultures with the natural D-dCyd as substrate for dCK, thus reducing the incorporation of exogenous [3H]dCyd into DNA. L-dCyd had no effect on the pool of dTTP deriving from the salvage or from the de novo synthesis, does not inhibit short term RNA and protein syntheses, and shows little or no cytotoxicity. Our results indicate a catalytic similarity between human dCK and herpetic thymidine kinases, enzymes that also lack stereospecificity. This functional analogy underlines the potential role of dCK as activator of L-deoxycytidine analogs as antiviral and antineoplastic agents and lends support to the hypothesis that herpesvirus thymidine kinase might have evolved from a captured cellular dCK gene, developing the ability to phosphorylate thymidine and retaining that to phosphorylate deoxycytidine.
Mol Pharmacol 1997 Jan
PMID:Lack of enantiospecificity of human 2'-deoxycytidine kinase: relevance for the activation of beta-L-deoxycytidine analogs as antineoplastic and antiviral agents. 901 55

Nickel has been shown to inhibit DNA repair in a way that may play a role in its toxicity. Since nickel treatment increases cellular reactive oxygen species (ROS), we have investigated the involvement of ROS in nickel inhibition of DNA repair. Inhibition of glutathione synthesis or catalase activity increased the enhancing effect of nickel on the cytotoxicity of ultraviolet (UV) light. Inhibition of catalase and glutathione peroxidase activities also enhanced the retardation effect of nickel on the rejoining of DNA strand breaks accumulated by hydroxyurea plus cytosine-beta-D-arabinofuranoside in UV-irradiated cells. Since DNA polymerization and ligation are involved in the DNA-break rejoining, we have investigated the effect of ROS on these two steps in an extract of Chinese hamster ovary cells. Nickel inhibition of the incorporation of (3H)dTTP into the DNase I-activated calf thymus DNA was stronger than the ligation of poly(dA) x oligo(dT), whereas H2O2 was more potent in inhibiting DNA ligation than DNA polymerization. Nickel, in the presence of H2O2, exhibited a synergistic inhibition on both DNA polymerization and ligation and caused protein fragmentation. In addition, glutathione could completely recover the inhibition by nickel or H2O2 alone but only partially recover the inhibition by nickel plus H2O2. Therefore, nickel may bind to DNA-repair enzymes and generate oxygen-free radicals to cause protein degradation in situ. This irreversible damage to the proteins involved in DNA repair, replication, recombination, and transcription could be important for the toxic effects of nickel.
Environ Mol Mutagen 1997
PMID:Reactive oxygen species are involved in nickel inhibition of DNA repair. 911 72

The effects of deoxynucleoside triphosphate (dNTP) imbalances on the fidelity of human immunodeficiency virus type 1 (HIV-1) replication were investigated. Using detergent permeabilized virions and biased dNTP concentrations different types of hypermutants were readily produced. However, the mutant spectrum was different from naturally occurring hypermutants demonstrating that the host cell may restrict variation. Using a genetic screen based on the blue/white beta-galactosidase complementation assay, G --> A hypermutants were recovered from HIV-infected thymidine treated U937 cells. Furthermore, hypermutants were recovered from 1 to 2% of resting or activated peripheral blood mononuclear cells indicating that small proportions of primary cells had distorted intracellular [dTTP] and [dCTP]. Such imbalances may underlie a proportion of somatic and germline point mutations and shape to some extent the evolution of mammalian and viral genomes.
J Mol Biol 1997 Jul 11
PMID:HIV genetic variation is directed and restricted by DNA precursor availability. 923 17


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