DNA and DNA precursors (deoxyribonucleotides) suffer damage by reactive oxygen/nitrogen species. They are important mutagens for organisms, due to their endogenous formation. Damaged DNA and nucleotides cause alterations of the genetic information by the mispairing properties of the damaged bases, such as 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) and 2-hydroxyadenine. Here, the author reviews the mutagenic potentials of damaged bases in DNA and of damaged DNA precursors formed by reactive oxygen/nitrogen species, focusing on the results obtained with synthetic oligonucleotides and 2'-deoxyribonucleoside 5'-triphosphates.

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.

The possibility that Escherichia coli MutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OHdATP), oxidized forms of dATP, but not (R)-8,5-cyclodATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OHdGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OHdGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2. However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.Endogenous oxidation of DNA and DNA precursors by reactive oxygen species appears to induce spontaneous mutations, aging, and various diseases, including cancer and neurodegeneration (1, 2). 8-OH-dGTP 1 is an oxidized form of dGTP and induces A:T to C:G transversions because it can pair with adenine as well as cytosine (3-6). It is known that the Escherichia coli MutT protein hydrolyzes 8-OH-dGTP to 8-hydroxydGMP (4). Because the mutation rate in a mutT-deficient strain increases up to 1000-fold as compared with the wild type (7), 8-OH-dGTP is considered to be a major source of spontaneous mutations caused by endogenous reactive oxygen species, and MutT appears to efficiently prevent the spontaneous occurrence of A:T to C:G transversion mutations. In human cells, the hMTH1 protein is considered to be a functional homologue of the E. coli MutT because the hMTH1 protein hydrolyzes 8-OH-dGTP in vitro and suppresses the mutator phenotype of E. coli mutT-deficient cells (8, 9).Recently, we found that 2-hydroxy-dAdo and 2-OH-dATP are produced efficiently by reactive oxygen species treatment of dAdo and dATP, respectively (10, 11). 2-OH-dATP specifically induces G:C to T:A transversion mutations and is more mutagenic than 8-OH-dGTP in vivo (5). Thus, 2-OH-dATP is thought to act as an endogenous mutagen in cells. However, the presence of a hydrolyzing activity for 2-OH-dATP has not been described. We supposed that the MutT and hMTH1 proteins may act on this mutagenic nucleotide, 2-OH-dATP. We report here that the hMTH1 protein, which is known as an 8-OHdGTPase, hydrolyzes 2-OH-dATP more efficiently than 8-OHdGTP. In addition, hMTH1 also hydrolyzed 8-OH-dATP, another oxidized form of dATP, as efficiently as 8-OH-dGTP. On the other han...

Liposomes are one of the most promising systems for selective cellular targeting via introduction of specific ligands for cell-surface receptors. After being taken up by the cells, these liposomes usually follow intracellular pathways of receptor-mediated endocytosis. Control of intracellular trafficking is required for optimized drug delivery. In this study, we elucidated the intracellular fate of transferrin-modified liposomes and succeeded in altering it by introducing the pH-sensitive fusogenic peptide, GALA (WEAALAEALAEALAEHLAEALAEALEALAA). Transferrins that are chemically attached to a liposomal surface (Tf-L) were internalized via receptor-mediated endocytosis more slowly than unmodified transferrins. In contrast to the recyclable nature of transferrin, liposome-attached transferrins together with encapsulated rhodamines were retained in vesicular compartments. When GALA was introduced into liposomal membranes using a cholesteryl moiety for anchoring (Chol-GALA), rhodamines were efficiently released and diffused into the cytosol. The addition of GALA to the Tf-L-containing medium or the encapsulation of GALA in Tf-L did not induce similar effects. These results clearly indicate that GALA must be present on the surface of liposomes to exert its function. In vitro energy transfer and dynamic light scattering experiments suggested that the endosomal escape of the encapsulates in Tf-L equipped with Chol-GALA can be attributed to pH-dependent membrane fusion. With GALA present on the surface, intracellular trafficking of liposomes after receptor-mediated endocytosis could be successfully controlled.