PubMed · 7350524
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A Travers. 1980-01-03. Why ppGpp?. https://doi.org/10.1038/283016a0
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Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.
Decauridylate containing exclusively a 2'-5' phospho-diester bond ([2'-5']U10) served as a template for the synthesis of oligoadenylates [oligo(A)s] from the 5'-phosphorimidazolide of 2'-5' diadenylate (ImpA-2'p5'A). Joining of [2'-5']U10and ImpA2'p5'A also took place in substantial amounts to yield long-chain oligoribonucleotides in the template-directed reaction. An unusual CD spectrum ascribed to helix formation between [2'-5']U10and [2'-5'](pA)2was observed under the same conditions as that of the template-directed reaction. The 3'-5' linked decauridylate ([3'-5']U10) also promoted the template-directed synthesis of oligo(A)s from ImpA2'p5'A, but more slowly compared with [2'-5']U10. The results indicate that short-chain RNA oligomers with a 2'-5' phosphodiester bond could lead to longer oligoribonucleotides by template-directed chain elongation.
In the present study the effect of ascorbate (0.8 mM)/iron (2.5 microM) on lipid and protein oxidation, in Synaptosomes isolated from rat brain cortex, was evaluated. Vitamin E, idebenone and reduced glutathione were used as free radicals scavengers, in order to analyze the mechanism involved in ascorbate/iron-induced oxidative stress. An increased formation of reactive oxygen species (ROS) in the cytosol and in the mitochondria was observed, in ascorbate/iron treated synaptosomes. Idebenone (50 microM) prevented the increased formation of ROS in both synaptosomal compartments, vitamin E (150 microM) protected partially this formation in mitochondria, whereas reduced glutathione (250 microM) (GSH) was ineffective. After ascorbate/iron treatment an increase in lipid peroxidation occurred as compared to control, which was completely inhibited by idebenone. A decrease in protein-SH content was also observed, and it was prevented by Vitamin E, idebenone and GSH. When synaptosomes were treated with ascorbate/iron the levels of GSH decreased, and the levels of oxidized glutathione (GSSG) increased as compared to controls under these conditions. Glutathione peroxidase activity was unchanged, whereas an inhibition of glutathione reductase activity was observed. These data suggest that the increased formation of free radicals in synaptosomes leads to lipid and protein oxidation, the role of the endogenous GSH being essential to protect protein thiol-groups against oxidative damage in order to maintain enzyme activity.