Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase

3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N6-ethenoadenine (ɛA) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only ɛA DNA glycosylase in liver, testes, and kidney; another ɛA DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag −/− mice are alkylation sensitive, indicating that Aag −/− mice may be similarly sensitive.

Inverse radiation dose-rate effects on somatic and germ-line mutations and DNA damage rates

The mutagenic effect of low linear energy transfer ionizing radiation is reduced for a given dose as the dose rate (DR) is reduced to a low level, a phenomenon known as the direct DR effect. Our reanalysis of published data shows that for both somatic and germ-line mutations there is an opposite, inverse DR effect, with reduction from low to very low DR, the overall dependence of induced mutations being parabolically related to DR, with a minimum in the range of 0.1 to 1.0 cGy/min (rule 1). This general pattern can be attributed to an optimal induction of error-free DNA repair in a DR region of minimal mutability (MMDR region). The diminished activation of repair at very low DRs may reflect a low ratio of induced (“signal”) to spontaneous background DNA damage (“noise”). Because two common DNA lesions, 8-oxoguanine and thymine glycol, were already known to activate repair in irradiated mammalian cells, we estimated how their rates of production are altered upon radiation exposure in the MMDR region. For these and other abundant lesions (abasic sites and single-strand breaks), the DNA damage rate increment in the MMDR region is in the range of 10% to 100% (rule 2). These estimates suggest a genetically programmed optimatization of response to radiation in the MMDR region.

Response of human DNA polymerase ι to DNA lesions

Lesion bypass is an important mechanism to overcome replication blockage by DNA damage. Translesion synthesis requires a DNA polymerase (Pol). Human Pol ι encoded by the RAD30B gene is a recently identified DNA polymerase that shares sequence similarity to Pol η. To investigate whether human Pol ι plays a role in lesion bypass we examined the response of this polymerase to several types of DNA damage in vitro. Surprisingly, 8-oxoguanine significantly blocked human Pol ι. Nevertheless, translesion DNA synthesis opposite 8-oxoguanine was observed with increasing concentrations of purified human Pol ι, resulting in predominant C and less frequent A incorporation opposite the lesion. Opposite a template abasic site human Pol ι efficiently incorporated a G, less frequently a T and even less frequently an A. Opposite an AAF-adducted guanine, human Pol ι was able to incorporate predominantly a C. In both cases, however, further DNA synthesis was not observed. Purified human Pol ι responded to a template TT (6–4) photoproduct by inserting predominantly an A opposite the 3′ T of the lesion before aborting DNA synthesis. In contrast, human Pol ι was largely unresponsive to a template TT cis-syn cyclobutane dimer. These results suggest a role for human Pol ι in DNA lesion bypass.

Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch

Adenine-DNA glycosylase MutY of Escherichia coli catalyzes the cleavage of adenine when mismatched with 7,8-dihydro-8-oxoguanine (GO), an oxidatively damaged base. The biological outcome is the prevention of C/G→A/T transversions. The molecular mechanism of base excision repair (BER) of A/GO in mammals is not well understood. In this study we report stimulation of mammalian adenine-DNA glycosylase activity by apurinic/apyrimidinic (AP) endonuclease using murine homolog of MutY (Myh) and human AP endonuclease (Ape1), which shares 94% amino acid identity with its murine homolog Apex. After removal of adenine by the Myh glycosylase activity, intact AP DNA remains due to lack of an efficient Myh AP lyase activity. The study of wild-type Ape1 and its catalytic mutant H309N demonstrates that Ape1 catalytic activity is required for formation of cleaved AP DNA. It also appears that Ape1 stimulates Myh glycosylase activity by increasing formation of the Myh–DNA complex. This stimulation is independent of the catalytic activity of Ape1. Consequently, Ape1 preserves the Myh preference for A/GO over A/G and improves overall glycosylase efficiency. Our study suggests that protein–protein interactions may occur in vivo to achieve efficient BER of A/GO.

A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

Werner syndrome (WS) is a premature aging disorder where the affected individuals appear much older than their chronological age. The single gene that is defective in WS encodes a protein (WRN) that has ATPase, helicase and 3′→5′ exonuclease activities. Our laboratory has recently uncovered a physical and functional interaction between WRN and the Ku heterodimer complex that functions in double-strand break repair and V(D)J recombination. Importantly, Ku specifically stimulates the exonuclease activity of WRN. We now report that Ku enables the Werner exonuclease to digest through regions of DNA containing 8-oxoadenine and 8-oxoguanine modifications, lesions that have previously been shown to block the exonuclease activity of WRN alone. These results indicate that Ku significantly alters the exonuclease function of WRN and suggest that the two proteins function concomitantly in a DNA damage processing pathway. In support of this notion we also observed co-localization of WRN and Ku, particularly after DNA damaging treatments.

Oxidative DNA damage and senescence of human diploid fibroblast cells.

Human diploid fibroblast cells cease growth in culture after a finite number of population doublings. To address the cause of growth cessation in senescent IMR-90 human fibroblast cells, we determined the level of oxidative DNA damage by using 8-oxoguanine excised from DNA and 8-oxo-2'-deoxyguanosine in DNA as markers. Senescent cells excise from DNA four times more 8-oxoguanine per day than do early-passage young cells. The steady-state level of 8-oxo-2'-deoxyguanosine in DNA is approximately 35% higher in senescent cells than in young cells. Measurement of protein carbonyls shows that senescent cells did not appear to have elevated protein oxidation. To reduce the level of oxidative damage, we cultured cells under a more physiological O2 concentration (3%) and compared the replicative life span to the cells cultured at the O2 concentration of air (20%). We found that cells grown under 3% O2 achieved 50% more population doublings during their lifetime. Such an extension of life span resulted from the delayed onset of senescence and elevation of growth rate and saturation density of cells at all passages. The spin-trapping agent alpha-phenyl-t-butyl nitrone (PBN), which can act as an antioxidant, also effectively delayed senescence and rejuvenated near senescent cells. The effect is dose-dependent and is most pronounced for cells at the stage just before entry into senescence. Our data support the hypothesis that oxidative DNA damage contributes to replicative cessation in human diploid fibroblast cells.

Biomarkers

Introduction – Why do we need ‘biomarkers? Biomarkers of protein oxidation Introduction Major issues/questions Protein carbonyl biomarkers Biochemistry Methods of measurement Storage, stability and limitations in use Protein thiol biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Aliphatic amino acid biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Oxidised Tryptophan Biomarkers Biochemistry Method of measurement Storage, stability and limitations on use Oxidised tyrosine biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Formation of neoepitopes on oxidised proteins Validation of assays for protein oxidation biomarkers Relationship of protein oxidation to disease Modulation of protein oxidation biomarkers by antioxidants Future perspectives Introduction to lipid peroxidation biomarkers Introduction: biochemistry of lipid peroxidation Malondialdehyde Methods of measurement Storage, stability and limitations on use Conjugated dienes Method of measurement Storage, stability and limitations of use LDL lag phase Method of measurement Storage, stability and limitations of use Hydrocarbon gases Biochemistry Method of measurement Storage, stability and limitations on use Lipofuscin Biochemistry Method of measurement Storage, stability and limitation on use Lipid peroxides Biochemistry Method of measurement Storage, stability and limitations on use Isoprostanes Biochemistry Method of measurement Storage, stability and limitations on use Possible new biomarkers of lipid oxidation Relationship of lipid peroxidation to disease Modulation of lipid peroxidation biomarkers by antioxidants Functional consequences of lipid peroxidation Contribution of dietary intake to lipid peroxidation products Biomarkers of DNA oxidation Introduction Confounding factors Units and terminology Nuclear and mitochondrial DNA damage Lymphocytes as surrogate tissues Measurement of DNA damage with the comet assay Practical details Storage, stability, and limitations of the assay Measurement of DNA base oxidation by HPLC Practical details Storage, stability and limitations of the method Measurement of DNA base oxidation by GC–MS Biochemistry of 8-oxoguanine, adenine and fapy derivatives Methods of measurement Storage, stability and limitations of the method Analysis of guanine oxidation products in urine Method of measurement Limitations and criticisms Immunochemical methods Methods of measurement Storage, stability, and limitations of the assay 32P post-labelling Method of measurement Limitations and criticisms Validation of assays for DNA oxidation Oxo-dGuo in lymphocyte DNA Urinary measurements DNA–aldehyde adducts Biochemistry Method of measurement Products of reactive nitrogen species Endpoints arising from oxidative DNA damage Mutations Chromosome aberrations Micronuclei Site-specific DNA damage Relationship of DNA oxidation to disease Modulation of DNA oxidation biomarkers by antioxidants Direct and indirect effects of oxidative stress: measures of total oxidant/antioxidant levels Visualisation of cellular oxidants Biochemistry: histochemical detection of ROS Method of measurement Limitations, storage and stability Measurement of hydrogen peroxide Biochemistry Methods of measurement Storage, stability and limitations on use Measurement of the ratio of antioxidant/oxidised antioxidant Biochemistry Method of measurement Storage, stability and limitations on use Total antioxidant capacity Biochemistry Terminology Methods of measurement Storage, stability and limitations on use Validation of assays for direct oxidant and antioxidant biomarkers Relationship of oxidant/antioxidant measurement to disease Modulation of oxidant/antioxidant biomarkers by dietary antioxidants Induction of genes in response to oxidative stress Background Measurement of antioxidant responsive genes and proteins Effects of antioxidant intake on the activity of antioxidant enzymes

Avaliação do efeito da inibição do reparo de sítios abásicos na resposta inflamatória celular

Base excision repair (BER) proteins has been associated with functions beyond DNA repair. Apurynic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein involved in a plethora of cellular activities, such as redox activation of transcription factors, RNA processing and DNA repair. Some studies have described the action of the protein 8-oxoguanine (OGG1) in correcting oxidized lesions in promoters as a step in the transcription of pro-inflammatory cytokines. Despite being especially important in redox activation of transcription factors such as nuclear factor κB (NF-κB) and AP- 1, the repair activity of APE1 has not yet been associated with the inflammatory response. In this study, experimental and bioinformatic analysis approaches have been used to investigate the relationship between inhibition of the repair of abasic sites in DNA by MX, a synthetic molecule designed to inhibt the repair activity of APE1, and the modulation of the inflammatory response. The results showed that treatment of monocytes with lipopolysaccharide (LPS) and MX reduced the expression of cytokines, chemokines and toll-like receptors, and negatively regulated biological immune processes, as macrophages activation, and NF-κB and tumor necrosis factor (TNF-α) and interferon pathways, without inducing cell death. The transcriptomic analysis suggests that LPS/MX treatment induces mitochondrial dysfunction, endoplasmic reticulum stress and activation of autophagy pathways, probably activated by impairment of cellular energy and/or the accumulation of nuclear and mitochondria DNA damage. Additionally, it is proposed that the repair activity of APE1 is required for transcription of inflammatory genes by interaction with abasic sites at specific promoters and recruitment of transcriptional complexes during inflammatory signaling. This work presents a new perspective on the interactions between the BER activity and the modulation of inflammatory response, and suggests a new activity for APE1 protein as modulator of the immune response in a redox-independent manner.

Oxidative DNA Damage Modulates Trinucleotide Repeat Instability Via DNA Base Excision Repair

Trinucleotide repeat (TNR) expansion is the cause of more than 40 types of human neurodegenerative diseases such as Huntington’s disease. Recent studies have linked TNR expansion with oxidative DNA damage and base excision repair (BER). In this research, we provided the first evidence that oxidative DNA damage can induce CAG repeat deletion/contraction via BER. We found that BER of an oxidized DNA base lesion, 8-oxoguanine in a CAG repeat tract, resulted in the formation of a CTG hairpin at the template strand. DNA polymerase β (pol b) then skipped over the hairpin creating a 5’-flap that was cleaved by flap endonuclease 1 (FEN1) leading to CAG repeat deletion. To further investigate whether BER may help to shorten an expanded TNR tract, we examined BER in a CAG repeat hairpin loop. We found that 8-oxoguanine DNA glycosylase removed the oxidized base located in the loop region of the hairpin leaving an abasic site. Apurinic/apyrimidinic (AP) endonuclease 1 then incised the 5’-end of the abasic site leaving a nick in the loop. This further converted the hairpin into an intermediate with a 3’-flap and a 5’-flap. As a 5’-3’ endonuclease, FEN1 cleaved the 5’-flap, whereas a 3’-5’ endonuclease, Mus81/Eme1, removed the 3’-flap. The coordination between FEN1 and Mus81/Eme1 ultimately resulted in removal of a CAG repeat hairpin attenuating or preventing TNR expansion. To further explore if pol β bypass of an oxidized base lesion, 5’,8-cyclodeoxyadenosine, may affect TNR instability, we examined pol β DNA synthesis in bypassing this base lesion and found that the lesion preferentially induced TNR deletion during BER and Okazaki fragment maturation. The repeat deletion was mediated by the formation of a loop in the template strand induced specifically by the damage. Pol β then skipped over the loop structure creating a 5’-flap that was efficiently removed by FEN1 leading to repeat deletion. Our study demonstrates that pol β-mediated BER plays an important role in mediating TNR deletion and removing a TNR hairpin to prevent TNR expansion. Our research provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

Effects of physical exercise training in DNA damage and repair activity in humans with different genetic polymorphisms ofhOGG1(Ser326Cys)

The main purpose of this pilot study was to investigate the possible influence of genetic polymorphisms of the hOGG1 (Ser326Cys) gene in DNA damage and repair activity by 8-oxoguanine DNA glycosylase 1 (OGG1 enzyme) in response to 16 weeks of combined physical exercise training. Thirty-two healthy Caucasian men (40-74 years old) were enrolled in this study. All the subjects were submitted to a training of 16 weeks of combined physical exercise. The subjects with Ser/Ser genotype were considered as wild-type group (WTG), and Ser/Cys and Cys/Cys genotype were analysed together as mutant group (MG). We used comet assay in conjunction with formamidopyrimidine DNA glycoslyase (FPG) to analyse both strand breaks and FPG-sensitive sites. DNA repair activity were also analysed with the comet assay technique. Our results showed no differences between DNA damage (both strand breaks and FPG-sensitive sites) and repair activity (OGG1) between genotype groups (in the pre-training condition). Regarding the possible influence of genotype in the response to 16 weeks of physical exercise training, the results revealed a decrease in DNA strand breaks in both groups, a decrease in FPG-sensitive sites and an increase in total antioxidant capacity in the WTG, but no changes were found in MG. No significant changes in DNA repair activity was observed in both genotype groups with physical exercise training. This preliminary study suggests the possibility of different responses in DNA damage to the physical exercise training, considering the hOGG1 Ser326Cys polymorphism.

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes: Part 14. The infrared and Raman characteristics of phthalocyanine in “pinwheel-like” homoleptic bis[1,8,15,22-tetrakis(3-pentyloxy)phthalocyaninato] rare earth(III) double-decker complexes

The infrared (IR) spectroscopic data and Raman spectroscopic properties for a series of 13 “pinwheel-like” homoleptic bis(phthalocyaninato) rare earth complexes M[Pc(α-OC5H11)4]2 [M = Y and Pr–Lu except Pm; H2Pc(α-OC5H11)4 = 1,8,15,22-tetrakis(3-pentyloxy)phthalocyanine] have been collected and comparatively studied. Both the IR and Raman spectra for M[Pc(α-OC5H11)4]2 are more complicated than those of homoleptic bis(phthalocyaninato) rare earth analogues, namely M(Pc)2 and M[Pc(OC8H17)8]2, but resemble (for IR) or are a bit more complicated (for Raman) than those of heteroleptic counterparts M(Pc)[Pc(α-OC5H11)4], revealing the decreased molecular symmetry of these double-decker compounds, namely S8. Except for the obvious splitting of the isoindole breathing band at 1110–1123 cm−1, the IR spectra of M[Pc(α-OC5H11)4]2 are quite similar to those of corresponding M(Pc)[Pc(α-OC5H11)4] and therefore are similarly assigned. With laser excitation at 633 nm, Raman bands derived from isoindole ring and aza stretchings in the range of 1300–1600 cm−1 are selectively intensified. The IR spectra reveal that the frequencies of pyrrole stretching and pyrrole stretching coupled with the symmetrical CH bending of –CH3 groups are sensitive to the rare earth ionic size, while the Raman technique shows that the bands due to the isoindole stretchings and the coupled pyrrole and aza stretchings are similarly affected. Nevertheless, the phthalocyanine monoanion radical Pc′− IR marker band of bis(phthalocyaninato) complexes involving the same rare earth ion is found to shift to lower energy in the order M(Pc)2 > M(Pc)[Pc(α-OC5H11)4] > M[Pc(α-OC5H11)4]2, revealing the weakened π–π interaction between the two phthalocyanine rings in the same order.

8-Ammonionaphthalene-2-sulfonate monohydrate : the zwitterionic hydrate of 1,7-Cleve's acid

The structure of 8-amino-2-naphthalenesulfonic acid monohydrate (1,7-Cleve's acid hydrate), C10H9NO3S.H2O, shows the presence of a sulfonate-aminium group zwitterion, both groups and the water molecule of solvation giving cyclic R3/3(8) intermolecular hydrogen-bonding interactions forming chains which extend down a axis of the unit cell. Additional peripheral associations, including weak aromatic ring pi-pi interactions [centroid-centroid distance 3.6299(15)A], result in a two-dimensional sheet structure.

Raman spectroscopic study of the uranyl sulphate mineral jáchymovite (UO2)8(SO4)(OH)14•13H2O

Raman spectra of jáchymovite, (UO2)8(SO4)(OH)14•13H2O, were studied, complemented with infrared spectra, and compared with published Raman and infrared spectra of uranopilite, [(UO2)6(SO4)O2(OH)6(H2O)6] •6H2O. Bands related to the stretching and bending vibrations of (UO2)2+, (SO4)2-, (OH)- and water molecules were assigned. U-O bond lengths in uranyl and O-H…O hydrogen bond lengths were calculated from the Raman and infrared spectra.