DNA damage by the sulfate radical anion: hydrogen abstraction from the sugar moiety versus one-electron oxidation of guanine

The products of oxidative damage to double-stranded (ds) DNA initiated by photolytically generated sulfate radical anions SO₄^?? were analyzed using reverse-phase (RP) high-performance liquid chromatography (HPLC). Relative efficiencies of two major pathways were compared: production of 8-oxoguanine (8oxoG) and hydrogen abstraction from the DNA 2-deoxyribose moiety (dR) at C1,′ C4,′ and C5′ positions. The formation of 8oxoG was found to account for 87% of all quantified lesions at low illumination doses. The concentration of 8oxoG quickly reaches a steady state at about one 8oxoG per 100 base pairs due to further oxidation of its products. It was found that another guanine oxidation product identified as 2-amino-5-(2′-alkylamino)-4H-imidazol-4-one (X) was released in significant quantities from its tentative precursor 2-amino-5-[(2′-deoxy-β-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) upon treatment with primary amines in neutral solutions. The linear dose dependence of X release points to the formation of dIz directly from guanine and not through oxidation of 8oxoG. The damage to dR was found to account for about 13% of the total damage, with majority of lesions (33%) originating from the C4′ oxidation. The contribution of C1′ oxidation also turned out to be significant (17% of all dR damages) despite of the steric problems associated with the abstraction of the C1′-hydrogen. However, no evidence of base-to-sugar free valence transfer as a possible alternative to direct hydrogen abstraction at C1′ was found.     

XPA protein as a limiting factor for nucleotide excision repair and UV sensitivity in human cells

Nucleotide excision repair (NER) acts on a variety of DNA lesions, including damage induced by many chemotherapeutic drugs. Cancer therapy with such drugs might be improved by reducing the NER capacity of tumors. It is not known, however to what extent any individual NER protein is rate-limiting for any step of the repair reaction. We studied sensitivity to UV radiation and repair of DNA damage with regard to XPA, one of the core factors in the NER incision complex. About 150,000-200,000 molecules of XPA protein are present in NER proficient human cell lines, and no XPA protein in the XP-A cell line XP12RO. Transfected XP12RO cell lines expressing 50,000 or more XPA molecules/cell showed UV resistance similar to normal cells. Suppression of XPA protein to approximately 10,000 molecules/cell in a Tet-regulatable system modestly but significantly increased sensitivity to UV irradiation. No removal of cyclobutane pyrimidine dimers was detected in the SV40 immortalized cell lines tested. Repair proficient WI38-VA fibroblasts and transfected XP-A cells expressing 150,000 molecules of XPA/cell removed (6-4) photoproducts from the genome with a half-life of 1h. Cells in which XPA protein was reduced to about 10,000 molecules/cell removed (6-4) photoproducts more slowly, with a half-life of 3h. A reduced rate of repair of (6-4) photoproducts thus results in increased cellular sensitivity towards UV irradiation. These data indicate that XPA levels must be reduced to <10% of that present in a normal cell to render XPA a limiting factor for NER and consequent cellular sensitivity. To inhibit NER, it may be more effective to interfere with XPA protein function, rather than reducing XPA protein levels.     

The release of 5-methylene-2-furanone from irradiated DNA catalyzed by cationic polyamines and divalent metal cations

Release of 5-methylene-2-furanone (5-MF), a characteristic marker of DNA deoxyribose oxidative damage at the C1' position, was observed in significant quantities from X-irradiated DNA. This observation, which held for DNA irradiated either in aqueous solution or as a film, requires postirradiation treatment at 90 degrees C in the presence of polyamines and divalent metal cations at biological pH. The 5-MF product was quantified by using reverse-phase HPLC. The radiation chemical yield of 5-MF comprised more than 30% of the yield of total unaltered base release. Polylysine, spermine and Be(II) showed the strongest catalytic effect on 5-MF release, while Zn(II), Cu(II), Ni(II), putrescine and Mg(II) were substantially less efficient. We have hypothesized that the 5-MF release from irradiated DNA occurs through catalytic decomposition of the 2'-deoxyribonolactone (dL) precursor through two consecutive beta- and delta-phosphate elimination reactions. A stepwise character of the process was indicated by the S-shaped time course of 5-MF accumulation. If dL proves to be the precursor to 5-MF formation, it would then follow that dL is a very important lesion generated in DNA by ionizing radiation.     

Effect of Anakinra on the Gene Expression of Receptors Activated by the Peroxisome Proliferator in the Rat Brain in the Lithium Pilocarpine Model of Epilepsy

Journal of Evolutionary Biochemistry and Physiology - The role of neuroinflammation in the mechanisms of epileptogenesis has been widely discussed in recent years. One of the factors influencing...     

Effects of mutations in the N terminal region of the yeast G protein α-subunit Gpa1p on signaling by pheromone receptors

The sites and modes of interaction between G protein-coupled receptors and their cognate heterotrimeric G proteins remain poorly defined. The C-terminus of the G subunit is the best established site of contact of G proteins with receptors, but structural analyses and crosslinking studies suggest the possibility of interactions at the N-terminus of G as well. We screened for mutations in the N-terminal region of the G subunit encoded by the yeast GPA1 gene that specifically affect the ability of the G protein to be activated by the yeast -mating factor receptor. The screen led to identification of substitutions of glutamine or proline for Leu18 of Gpa1p that reduce the response to the pheromones -factor and a-factor without affecting cellular levels of the subunit or its ability to interact with and subunits. The mutations do not appear to affect the intrinsic ability of the G protein to be converted to the activated state. The low yield of different mutations with this phenotype indicates either that the N-terminal segment of the yeast G subunit does not undergo extensive interactions with the -factor receptor, or that this region can not be altered without detrimental effects upon the formation of G protein trimers.Communicated by D. Y. Thomas     

Structural characterization of human RPA sequential binding to single-stranded DNA using ssDNA as a molecular ruler

Human replication protein A (RPA), a heterotrimer composed of RPA70, RPA32, and RPA14 subunits, contains four single-stranded DNA (ssDNA) binding domains (DBD): DBD-A, DBD-B, and DBD-C in RPA70 and DBD-D in RPA32. Although crystallographic or NMR structures of these DBDs and a trimerization core have been determined, the structure of the full length of RPA or the RPA-ssDNA complex remains unknown. In this article, we have examined the structural features of RPA interaction with ssDNA by fluorescence spectroscopy. Using a set of oligonucleotides (dT) with varying lengths as a molecular ruler and also as the substrate, we have determined at single-nucleotide resolution the relative positions of the ssDNA with interacting intrinsic tryptophans of RPA. Our results revealed that Trp528 in DBD-C and Trp107 in DBD-D contact ssDNA at the 16th and 24th nucleotides (nt) from the 5'-end of the substrate, respectively. Evaluation of the relative spatial arrangement of RPA domains in the RPA-ssDNA complex suggested that DBD-B and DBD-C are spaced by about 4 nt ( approximately 19 A) apart, whereas DBD-C and DBD-D are spaced by about 7 nt ( approximately 34 A). On the basis of these geometric constraints, a global structure model for the binding of the major RPA DBDs to ssDNA was proposed.     

Reductively activated cleavage of DNA mediated by o, o'-diphenylenehalonium compounds

o,o'-Diphenylenehalonium (DPH) cations represent a novel class of DNA-affinic compounds characterized by binding constants within the range of 10(5)-10(6) M(-1). The maximum binding capacity of 2-2.5 base pairs per DPH cation and about 30% hypochromic reduction in the optical absorption of DPH cations upon binding to DNA suggest intercalation as a likely binding mode. In a DNA-bound form, DPH cations induce strand breaks upon reduction by radiation-produced electrons in aqueous solutions. In keeping with this mechanism, the cleavage is strongly inhibited by oxygen and is not affected by OH radical scavengers in the bulk. The yields of DPH-mediated base release significantly exceed the yield of base release caused by hydroxyl radical (in the absence of scavenger) in anoxic solutions. The yields are weakly dependent on DNA loading within the range from 5 to 50 base pairs per intercalator, which indicates the ability of excess electrons in DNA to react with a scavenger separated by tens of base pairs from the electron attachment site. The question regarding the mechanism by which the distant reactants reach each other in DNA remains unanswered, although it most likely involves electron hopping rather than a single-step long-distance tunneling. The latter conclusion is based on our finding that the electron affinity of DPH cations does not affect their properties as electron scavengers in DNA as would be expected if the direct long-distance tunneling is involved.     

Comparative insecticidal acitvity of sumithrin and neopinamine

The examination of insecticidal properties of the new pyrethroid--sumithrin--showed this compound in contact action to be a highly effective insecticide, superior in activity to neopinamine. The intestinal action of sumithrin is less pronounced in comparison with neopinamine. Sumithrin is more selective in its effect on insects as compared with neopinamine. In the form of earosols, sumithrin and its mixtures with DDVP are less active than compositions based on neopinamine.