Structural Insights into the Specificity of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Family X DNA Polymerases

8-oxo-guanine (8OG) is a common base lesion, generated by reactive oxygen species, which has been associated with human diseases such as cancer, aging-related neurodegenerative disorders and atherosclerosis. 8OG is highly mutagenic, due to its dual-coding potential it can pair both with adenine or cytidine. Therefore, it creates a challenge for DNA polymerases striving to correctly replicate and/or repair genomic or mitochondrial DNA. Numerous structural studies provide insights into the mechanistic basis of the specificity of 8OG bypass by DNA polymerases from different families. Here, we focus on how repair polymerases from Family X (Pols β, λ and µ) engage DNA substrates containing the oxidized guanine. We review structures of binary and ternary complexes for the three polymerases, which represent distinct steps in their catalytic cycles—the binding of the DNA substrate and the incoming nucleotide, followed by its insertion and extension. At each of these steps, the polymerase may favor or exclude the correct C or incorrect A, affecting the final outcome, which varies depending on the enzyme.

Associations between physical activity, sedentary behavior, and urinary oxidized guanine in colorectal cancer patients: results from the ColoCare Study

To determine associations between physical activity (PA), sedentary behavior (SB), and oxidative stress in colorectal cancer patients, ColoCare Study participants in Germany wore an accelerometer 6 and/or 12 months after surgery. Spearman partial correlations were used to assess associations between PA and urinary concentrations of oxidized guanine, a validated marker of oxidative stress. There were no significant associations between PA or SB and oxidized guanine in n = 76 measurements (ng/mg creatinine; r = 0.03, p = 0.76 for PA, r = –0.05, p = 0.69 for SB). Novelty Objectively measured PA was not associated with a marker of oxidative stress in colorectal cancer patients.

Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization

To investigate the role of oxidative stress–induced DNA damage and mutagenesis in cellular senescence and immortalization, here we profiled spontaneous and methylene blue plus light–induced mutations in the cII gene from λ phage in transgenic mouse embryonic fibroblasts during the transition from primary culture through senescence and immortalization. Consistent with detection of characteristic oxidized guanine lesions (8-oxodG) in the treated cells, we observed significantly increased relative cII mutant frequency in the treated pre-senescent cells which was augmented in their immortalized counterparts. The predominant mutation type in the treated pre-senescent cells was G:C→T:A transversion, whose frequency was intensified in the treated immortalized cells. Conversely, the prevailing mutation type in the treated immortalized cells was A:T→C:G transversion, with a unique sequence-context specificity, i.e. flanking purines at the 5′ end of the mutated nucleotide. This mutation type was also enriched in the treated pre-senescent cells, although to a lower extent. The signature mutation of G:C→T:A transversions in the treated cells accorded with the well-established translesion synthesis bypass caused by 8-oxodG, and the hallmark A:T→C:G transversions conformed to the known replication errors because of oxidized guanine nucleosides (8-OHdGTPs). The distinctive features of photosensitization-induced mutagenesis in the immortalized cells, which were present at attenuated levels, in spontaneously immortalized cells provide insights into the role of oxidative stress in senescence bypass and immortalization. Our results have important implications for cancer biology because oxidized purines in the nucleoside pool can significantly contribute to genetic instability in DNA mismatch repair–defective human tumors.

Role of Mfd and GreA in Bacillus subtilis Base Excision Repair-Dependent Stationary-Phase Mutagenesis

ABSTRACT We report that the absence of an oxidized guanine (GO) system or the apurinic/apyrimidinic (AP) endonucleases Nfo, ExoA, and Nth promoted stress-associated mutagenesis (SAM) in Bacillus subtilis YB955 (hisC952 metB5 leuC427). Moreover, MutY-promoted SAM was Mfd dependent, suggesting that transcriptional transactions over nonbulky DNA lesions promoted error-prone repair. Here, we inquired whether Mfd and GreA, which control transcription-coupled repair and transcription fidelity, influence the mutagenic events occurring in nutritionally stressed B. subtilis YB955 cells deficient in the GO or AP endonuclease repair proteins. To this end, mfd and greA were disabled in genetic backgrounds defective in the GO and AP endonuclease repair proteins, and the strains were tested for growth-associated and stress-associated mutagenesis. The results revealed that disruption of mfd or greA abrogated the production of stress-associated amino acid revertants in the GO and nfo exoA nth strains, respectively. These results suggest that in nutritionally stressed B. subtilis cells, spontaneous nonbulky DNA lesions are processed in an error-prone manner with the participation of Mfd and GreA. In support of this notion, stationary-phase ΔytkD ΔmutM ΔmutY (referred to here as ΔGO) and Δnfo ΔexoA Δnth (referred to here as ΔAP) cells accumulated 8-oxoguanine (8-OxoG) lesions, which increased significantly following Mfd disruption. In contrast, during exponential growth, disruption of mfd or greA increased the production of His+, Met+, or Leu+ prototrophs in both DNA repair-deficient strains. Thus, in addition to unveiling a role for GreA in mutagenesis, our results suggest that Mfd and GreA promote or prevent mutagenic events driven by spontaneous genetic lesions during the life cycle of B. subtilis. IMPORTANCE In this paper, we report that spontaneous genetic lesions of an oxidative nature in growing and nutritionally stressed B. subtilis strain YB955 (hisC952 metB5 leuC427) cells drive Mfd- and GreA-dependent repair transactions. However, whereas Mfd and GreA elicit faithful repair events during growth to maintain genome fidelity, under starving conditions, both factors promote error-prone repair to produce genetic diversity, allowing B. subtilis to escape from growth-limiting conditions.

Photosensitized oxidation of 2′-deoxyguanosine 5′-monophosphate: mechanism of the competitive reactions and product characterization

We have identified five products containing oxidized guanine and another product containing an intact guanine moiety and a modified one.

Sirt3 protects mitochondrial DNA damage and blocks the development of doxorubicin-induced cardiomyopathy in mice

Doxorubicin (Doxo) is a chemotherapeutic drug widely used to treat variety of cancers. One of the most serious side effects of Doxo is its dose-dependent and delayed toxicity to the heart. Doxo is known to induce cardiac mitochondrial damage. Recently, the mitochondrial sirtuin SIRT3 has been shown to protect mitochondria from oxidative stress. Here we show that overexpression of SIRT3 protects the heart from toxicity of Doxo by preventing the drug-induced mitochondrial DNA (mtDNA) damage. Doxo treatment caused depletion of Sirt3 levels both in primary cultures of cardiomyocytes and in mouse hearts, which led to massive acetylation of mitochondrial proteins. Doxo-induced toxicity to cardiomyocytes was associated with increased reactive oxygen species (ROS) production, mitochondrial fragmentation, and cell death. Overexpression of SIRT3 helped to attenuate Doxo-induced ROS levels and cardiomyocyte death. Sirt3 knockout (Sirt3.KO) mice could not endure the full dose of Doxo treatment, developed exacerbated cardiac hypertrophy, and died during the course of treatment, whereas Sirt3 transgenic (Sirt3.tg) mice were protected against Doxo-induced cardiotoxicity. Along with Sirt3, we also observed a concomitant decrease in levels of oxoguanine-DNA glycosylase-1 (OGG1), a major DNA glycosylase that hydrolyzes oxidized-guanine (8-oxo-dG) to guanine. Depletion of OGG1 levels was associated with increased mtDNA damage. Sirt3.KO mice and Doxo-treated mice showed increased 8-oxo-dG adducts in DNA and corresponding increase in mtDNA damage, whereas, 8-oxo-dG adducts and mtDNA damage were markedly reduced in Sirt3 overexpressing transgenic mice hearts. These results thus demonstrated that Sirt3 activation protects the heart from Doxo-induced cardiotoxicity by maintaining OGG1 levels and protecting mitochondria from DNA damage.

Oxidative Stress Markers in Sputum

Although oxidative stress is thought to play a pivotal role in the pathogenesis of inflammatory airway diseases, its assessment in clinical practice remains elusive. In recent years, it has been conceptualized that oxidative stress markers in sputum should be employed to monitor oxidative processes in patients with asthma, chronic obstructive pulmonary disease (COPD), or cystic fibrosis (CF). In this review, the use of sputum-based oxidative markers was explored and potential clinical applications were considered. Among lipid peroxidation-derived products, 8-isoprostane and malondialdehyde have been the most frequently investigated, while nitrosothiols and nitrotyrosine may serve as markers of nitrosative stress. Several studies have showed higher levels of these products in patients with asthma, COPD, or CF compared to healthy subjects. Marker concentrations could be further increased during exacerbations and decreased along with recovery of these diseases. Measurement of oxidized guanine species and antioxidant enzymes in the sputum could be other approaches for assessing oxidative stress in pulmonary patients. Collectively, even though there are promising findings in this field, further clinical studies using more established detection techniques are needed to clearly show the benefit of these measurements in the follow-up of patients with inflammatory airway diseases.