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p66shc is a protein product of an mRNA isoform of SHC1 gene that has a pro-oxidant and pro-apoptotic activity and is implicated in the aging process. Mitochondria were suggested as a major source of the p66shc-mediated production of reactive oxygen species (ROS), although the underlying mechanisms are poorly understood. We studied effects of p66shc on oxidative stress induced by hydrogen peroxide or by serum deprivation in human colon carcinoma cell line RKO and in diploid human dermal fibroblasts (HDFs). An shRNA-mediated knockdown of p66shc suppressed and an overexpression of a recombinant p66shc stimulated the production of ROS in the both models. This effect was not detected in the mitochondrial DNA-depleted ρ0-RKO cells that do not have the mitochondrial electron transport chain (ETC). The p66shc-dependent accumulation of mitochondrial ROS was detected with HyPer-mito, a mitochondria-targeted fluorescent protein sensor for hydrogen peroxide. The fragmentation of mitochondria induced by mitochondrial ROS was significantly reduced in the p66shc deficient RKO cells. Mitochondria-targeted antioxidants SkQ1 and SkQR1 also decreased the oxidative stress induced by hydrogen peroxide or by serum deprivation. Together the data indicate that the p66shc-dependant ROS production during oxidative stress has mitochondrial origin in human normal and cancer cells. [ABSTRACT FROM AUTHOR]

DNA glycosylases are enzymes that initiate base excision repair, which removes damaged bases from cell DNA. Recent data demonstrate that some genetic variants of two human DNA glycosylases, MUTYH and OGG1, are associated with an increased risk of cancer. In addition, various DNA glycosylases are involved in protection from some neurodegenerative diseases, immune disorders, and virus infections. On the other hand, DNA glycosylases of pathogenic microorganisms help them to evade the host defense mechanisms. Thus, DNA glycosylases are considered to be both potential therapeutic agents and drug targets. [ABSTRACT FROM AUTHOR]

Many enzymes acting on specific rare lesions in DNA are suggested to search for their targets by facilitated one-dimensional diffusion. We have used a recently developed correlated cleavage assay to investigate whether this mechanism operates for Fpg and OGG1, two structurally unrelated DNA glycosylases that excise an important oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG), from DNA. Similar to a number of other DNA glycosylases or restriction endonucleases, Fpg and OGG1 processively excised 8-oxoG from pairs with cytosine at low salt concentrations, indicating that the lesion search likely proceeds by one-dimensional diffusion. At high salt concentrations, both enzymes switched to a distributive mode of lesion search. Correlated cleavage of abasic site-containing substrates proceeded in the same manner as cleavage of 8-oxoG. Interestingly, both Fpg and especially OGG1 demonstrated higher processivity if the substrate contained 8-oxoG·A pairs, against which these enzyme discriminate. Introduction of a nick into the substrate DNA did not decrease the extent of correlated cleavage, suggesting that the search probably involves hopping between adjacent positions on DNA rather than sliding along DNA. This was further supported by the observation that mutant forms of Fpg (Fpg-F110A and Fpg-F110W) with different sizes of the side chain of the amino acid residue inserted into DNA during scanning were both less processive than the wild-type enzyme. In conclusion, processive cleavage by Fpg and OGG1 does not correlate with their substrate specificity and under nearly physiological salt conditions may be replaced with the distributive mode of action. [ABSTRACT FROM AUTHOR]

An abundant oxidative lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), often directs the misincorporation of dAMP during replication. To prevent mutations, cells possess an enzymatic system for the removal of 8-oxoG. A key element of this system is 8-oxoguanine-DNA glycosylase (Fpg in bacteria, OGG1 in eukaryotes), which must excise 8-oxoG from 8-oxoG:C pairs but not from 8-oxoG:A. We investigated the influence of various factors, including ionic strength, the presence of Mg2+ and organic anions, polyamides, crowding agents and two small heterocyclic compounds (biotin and caffeine) on the activity and opposite-base specificity of Escherichia coli Fpg and human OGG1. The activity of both enzymes towards 8-oxoG:A decreased sharply with increasing salt and Mg2+ concentration, whereas the activity on 8-oxoG:C was much more stable, resulting in higher opposite-base specificity when salt and Mg2+ were at near-physiological concentrations. This tendency was observed with both Cl− and glutamate as the major anions in the reaction mixture. Kinetic and binding parameters for the processing of 8-oxoG:C and 8-oxoG:A by Fpg and OGG1 were determined under several different conditions. Polyamines, crowding agents, biotin and caffeine affected the activity and specificity of Fpg or OGG1 only marginally. We conclude that, in the intracellular environment, the specificity of Fpg and OGG1 for 8-oxoG:C versus 8-oxoG:A is mostly due to high ionic strength and Mg2+. [ABSTRACT FROM AUTHOR]