Flavia Barone

8-Oxoguanine incorporation into DNA repeats in vitro and mismatch recognition by MutSα

DNA 8-oxoguanine (8-oxoG) causes transversions and is also implicated in frameshifts. We previously identified the dNTP pool as a likely source of mutagenic DNA 8-oxoG and demonstrated that DNA mismatch repair prevented oxidation-related frameshifts in mononucleotide repeats. Here, we show that both Klenow fragment and DNA polymerase α can utilize 8-oxodGTP and incorporate the oxidized purine into model frameshift targets. Both polymerases incorporated 8-oxodGMP opposite C and A in repetitive DNA sequences and efficiently extended a terminal 8-oxoG. The human MutSα mismatch repair factor recognized DNA 8-oxoG efficiently in some contexts that resembled frameshift intermediates in the same C or A repeats. DNA 8-oxoG in other slipped/mispaired structures in the same repeats adopted configurations that prevented recognition by MutSα and by the OGG1 DNA glycosylase thereby rendering it invisible to DNA repair. These findings are consistent with a contribution of oxidative DNA damage to frameshifts. They also suggest how mismatch repair might reduce the burden of DNA 8-oxoG and prevent frameshift formation.

Comparative study of ZnO and TiO2 nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells

Abstract Despite human gastrointestinal exposure to nanoparticles (NPs), data on NPs toxicity in intestinal cells are quite scanty. In this study we evaluated the toxicity induced by zinc oxide (ZnO) and titanium dioxide (TiO2) NPs on Caco-2 cells. Only ZnO NPs produced significant cytotoxicity, evaluated by two different assays. The presence of foetal calf serum in culture medium significantly reduced ZnO NPs toxicity as well as ion leakage and NP-cell interaction. The two NPs increased the intracellular amount of reactive oxygen species (ROS) after 6 h treatment. However, only ZnO NPs increased ROS and induced IL-8 release both after 6 and 24 h. Experimental data indicate a main role of chemical composition and solubility in ZnO NPs toxicity. Moreover our results suggest a key role of oxidative stress in ZnO NPs cytotoxicity induction related both to ion leakage and to cell interaction with NPs in serum-free medium.

Role of MUTYH and MSH2 in the Control of Oxidative DNA Damage, Genetic Instability, and Tumorigenesis

Mismatch repair is the major pathway controlling genetic stability by removing mispairs caused by faulty replication and/or mismatches containing oxidized bases. Thus, inactivation of the Msh2 mismatch repair gene is associated with a mutator phenotype and increased cancer susceptibility. The base excision repair gene Mutyh is also involved in the maintenance of genomic integrity by repairing premutagenic lesions induced by oxidative DNA damage. Because evidence in bacteria suggested that Msh2 and Mutyh repair factors might have some overlapping functions, we investigated the biological consequences of their single and double inactivation in vitro and in vivo. Msh2(-/-) mouse embryo fibroblasts (MEF) showed a strong mutator phenotype at the hprt gene, whereas Mutyh inactivation was associated with a milder phenotype (2.9 x 10(-6) and 3.3 x 10(-7) mutation/cell/generation, respectively). The value of 2.7 x 10(-6) mutation/cell/generation in Msh2(-/-)Mutyh(-/-) MEFs did not differ significantly from Msh2(-/-) cells. When steady-state levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG) were measured in MEFs of different genotypes, single gene inactivation resulted in increases similar to those observed in doubly defective cells. In contrast, a synergistic accumulation of 8-oxoG was observed in several organs of Msh2(-/-)Mutyh(-/-) animals, suggesting that in vivo Msh2 and Mutyh provide separate repair functions and contribute independently to the control of oxidative DNA damage. Finally, a strong delay in lymphomagenesis was observed in Msh2(-/-)Mutyh(-/-) when compared with Msh2(-/-) animals. The immunophenotype of these tumors indicate that both genotypes develop B-cell lymphoblastic lymphomas displaying microsatellite instability. This suggests that a large fraction of the cancer-prone phenotype of Msh2(-/-) mice depends on Mutyh activity.

Different mechanisms are involved in oxidative DNA damage and genotoxicity induction by ZnO and TiO2 nanoparticles in human colon carcinoma cells.

In this work we investigated the genotoxicity of zinc oxide and titanium dioxide nanoparticles (ZnO NPs; TiO2 NPs) induced by oxidative stress on human colon carcinoma cells (Caco-2 cells). We measured free radical production in acellular conditions by Electron Paramagnetic Resonance technique and genotoxicity by micronucleus and Comet assays. Oxidative DNA damage was assessed by modified Comet assay and by measuring 8-oxodG steady state levels. The repair kinetics of DNA oxidation as well as the expression levels of hOGG1 were also analyzed. Even if both NPs were able to produce ROS in acellular conditions and to increase 8-oxodG levels in Caco-2 cells, only ZnO NPs resulted genotoxic inducing micronuclei and DNA damage. Furthermore, Caco-2 cells exposed to ZnO NPs were not able to repair the oxidative DNA damage that was efficiently repaired after TiO2 NPs treatment, through OGG1 involvement. These results indicate that the high oxidant environment caused by ZnO NPs in our cellular model can induce DNA damage and affect the repair pathways.

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay†

MUTYH‐associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8‐hydroxyguanine (8‐oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh−/− mouse defective cells. Several parameters, including accumulation of 8‐oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell‐based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild‐type protein. Our cell‐based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1–8, 2009.

Genotype-phenotype analysis of S326C OGG1 polymorphism: a risk factor for oxidative pathologies.

8-Oxoguanine DNA glycosylase (OGG) activity was measured by an in vitro assay in lymphocytes of healthy volunteers genotyped for various OGG1 polymorphisms. Only homozygous carriers of the polymorphic C326 allele showed a significantly lower OGG activity compared to the homozygous S326 genotype. The purified S326C OGG1 showed a decreased ability to complete the repair synthesis step in a base excision repair reaction reconstituted in vitro. The propensity of this variant to dimerize as well as its catalytic impairment were shown to be enhanced under oxidizing conditions. Mass spectrometry revealed that the extra cysteine of the variant protein is involved in disulfide bonds compatible with significant conformational changes and/or dimerization. We propose that the S326C OGG1 catalytic impairment and its susceptibility to dimerization and disulfide bond formation in an oxidizing environment all concur to decrease repair capacity. Consequently, the C326 homozygous carriers may be at increased risk of oxidative pathologies.

DNA, RNA and hybrid RNA-DNA oligomers of identical sequence: structural and dynamic differences.

A 27-mer sequence was synthesised as DNA duplex (DD), RNA duplex (RR), and RNA-DNA (RD) hybrid in order to characterise their structural and dynamic features. The hydrodynamic radius (Rh) and the rise (b) values of the three samples were consistent with the conformations predicted by CD analysis. The value of the torsional constant (alpha) of the samples containing RNA was approximately twice that of the DD sample and followed the order: DD < RD < RR. The same order was observed in the thermodynamic stability and in the reduction of the electrophoretic mobility. gamma-Ray footprinting analysis was carried out to resolve the individual strand conformation in the hybrid. The RNA strand preserved its conformation, while the DNA strand showed local deformations mainly at TA and TG steps.

Torsional constant of 27-mer DNA oligomers of different sequences

We have studied the torsional elastic constant (alpha) of short DNA (27mer) oligomers of various sequence by fluorescence polarization anysotropy (FPA) measurements. The lowest alpha values were found in samples with sequence rich in AA dinucleotides or containing the alternating d(A-T) x d(A-T) motif. The torsional rigidity of our DNA samples was compared to that calculated according to the current values of twist angle fluctuations derived for ten dinucleotide steps by recent analyses of DNA crystal structure database. The values of torsional rigidity derived from crystals are higher than our experimental ones, obtained by FPA analysis, suggesting that packing force in crystals may notably hinder the dinucleotide twist angle fluctuations that occur in solution. This behaviour is more evident for samples containing AA, TA and AT steps. In all the samples there is about a twofold change of the alpha value in the 10-40 degrees C range. An activation enthalpy (Delta H (#)) of about 17.4 kJ mol(-1), on average, was obtained for the temperature dependence of eight of the ten samples studied. A correlation with the stacking energy is discussed.

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.

Triple helix DNA oligomer melting measured by fluorescence polarization anisotropy.

Abstract A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 mer single strand sequence onto the complementary purinic sequence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10–90 °C. The Tm of the triplex, 37 °C, was well separated from the onset of duplex melting. When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the Tm of the triplex was shifted to approximately 32 °C and overlapped the melting of the duplex. We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and triplex samples to determine the hydrodynamic parameters in the temperature range 10–40 °C. The fluorescence lifetime of EB in the samples of double and triple stranded DNA is the same (21.3 ± 0.5 ns) at 20 °C, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal triplex, and nicked triplex samples were 10.7 ± 0.2, 12.2 ± 0.2, and 12.0 ± 0.2 Å. FPA measurements on normal triplex DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the triplex carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal triplex sample. The torsional rigidity of the triplex forms was found to be higher than that of the duplex form.