End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining

DNA polymerase μ is a member of the mammalian pol X family and reduces deletion during chromosome break repair by nonhomologous end joining (NHEJ). This biological role is linked to pol μ's ability to promote NHEJ of ends with noncomplementary 3′ overhangs, but questions remain regarding how it performs this role. We show here that synthesis by pol μ in this context is often rapid and, despite the absence of primer/template base-pairing, instructed by template. However, pol μ is both much less active and more prone to possible template independence in some contexts, including ends with overhangs longer than two nucleotides. Reduced activity on longer overhangs implies pol μ is less able to synthesize across longer gaps, arguing pol μ must bridge both sides of gaps between noncomplementary ends to be effective in NHEJ. Consistent with this argument, a pol μ mutant defective specifically on gapped substrates is also less active during NHEJ of noncomplementary ends both in vitro and in cells. Taken together, pol μ activity during NHEJ of noncomplementary ends can thus be primarily linked to pol μ's ability to work together with core NHEJ factors to bridge DNA ends and perform a template-dependent gap fill-in reaction

Novel ketone diet enhances physical and cognitive performance.

Ketone bodies are the most energy-efficient fuel and yield more ATP per mole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high-fat, low-carbohydrate diets has been used for the treatment of drug-refractory epilepsy and for neurodegenerative diseases, such as Parkinson's disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with (R)-3-hydroxybutyl (R)-3-hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a treadmill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palm oil (P < 0.05). Ketone-fed rats completed an 8-arm radial maze test 38% faster than did those on the other diets, making more correct decisions before making a mistake (P < 0.05). Isolated, perfused hearts from rats that were fed the ketone diet had greater free energy available from ATP hydrolysis during increased work than did hearts from rats on the other diets as shown by using [31P]-NMR spectroscopy. The novel ketone diet, therefore, improved physical performance and cognitive function in rats, and its energy-sparing properties suggest that it may help to treat a range of human conditions with metabolic abnormalities.-Murray, A. J., Knight, N. S., Cole, M. A., Cochlin, L. E., Carter, E., Tchabanenko, K., Pichulik, T., Gulston, M. K., Atherton, H. J., Schroeder, M. A., Deacon, R. M. J., Kashiwaya, Y., King, M. T., Pawlosky, R., Rawlins, J. N. P., Tyler, D. J., Griffin, J. L., Robertson, J., Veech, R. L., Clarke, K. Novel ketone diet enhances physical and cognitive performance.A.J.M. thanks the Research Councils UK for supporting his Academic Fellowship. This work was supported by the Defense Advanced Research Projects Agency.This is the final version of the article. It first appeared from FASEB at https://doi.org/10.1096/fj.201600773R

Identification of the Biotransformation Products of 2-Ethylhexyl 4-(N,N-Dimethylamino)benzoate

Nowadays, 2-ethylhexyl 4-(N,N-dimethylamino)benzoate (EDP) is one of the most widely used UV filters in sunscreen cosmetics and other cosmetic products. However, undesirable processes such as percutaneous absorption and biological activity have been attributed to this compound. The in vitro metabolism of EDP was elucidated in the present work. First of all, the phase I biotransformation was studied in rat liver microsomes and two metabolites, N,N-dimethyl-p-aminobenzoic acid (DMP) and N-monomethyl-p-aminobenzoic acid (MMP), were identified by GC-MS analysis. Secondly, the phase II metabolism was investigated by means of LC-MS. The investigated reactions were acetylation and glucuronidation working with rat liver cytosol and with both human and rat liver microsomes, respectively. Analogue studies with p-aminobenzoic acid (PABA) were carried out in order to compare the well established metabolic pathway of PABA with the unknown biotransformation of EDP. In addition, a method for the determination of EDP and its two phase I metabolites in human urine was developed. The methodology requires a solid-phase extraction prior to LC-MS analysis. The method is based on standard addition quantification and has been fully validated. The repeatability of the method, expressed as relative standard deviation, was in the range 3.4–7.4% and the limit of detection for all quantified analytes was in the low ng mL−1 range

DNA Dosimetry Assessment for Sunscreen Genotoxic Photoprotection

Background: Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. Methodology/Principal Findings: The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. Conclusions/Significance: The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.Natura Inovacao e Tecnologia de Produtos LTDA (Sao Paulo, Brazil)Natura Inovacao e Tecnologia de Produtos LTDA (Sao Paulo, Brazil)FAPESP (Sao Paulo, Brazil)FAPESP (Sao Paulo, Brazil)CNPq (Brasilia, Brazil)CNPq (Brasilia, Brazil)Natura Inovacao e Tecnologia de Produtos LTDANatura Inovacao e Tecnologia de Produtos LTD

Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation.

Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cells. Mutant hamster cells (xrs-5), deficient in non-homologous end joining (NHEJ), were irradiated at 37 degrees C to determine whether any additional double-strand breaks (DSBs) are formed during processing of gamma-radiation-induced DNA clustered damage sites. A class of non-DSB clustered DNA damage, corresponding to approximately 30% of the initial yield of DSBs, is converted into DSBs reflecting an artefact of preparation of genomic DNA for pulsed field gel electrophoresis. These clusters are removed within 4 min in both NHEJ-deficient and wild-type CHO cells. In xrs-5 cells, a proportion of non-DSB clustered DNA damage, representing approximately 10% of the total yield of non-DSB clustered DNA damage sites, are also converted into DSBs within approximately 30 min post-gamma but not post-alpha irradiation through cellular processing at 37 degrees C. That the majority of radiation-induced non-DSB clustered DNA damage sites are resistant to conversion into DSBs may be biologically significant at environmental levels of radiation exposure, as a non-DSB clustered damage site rather than a DSB, which only constitutes a minor proportion, is more likely to be induced in irradiated cells

Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation.

Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cells. Mutant hamster cells (xrs-5), deficient in non-homologous end joining (NHEJ), were irradiated at 37 degrees C to determine whether any additional double-strand breaks (DSBs) are formed during processing of gamma-radiation-induced DNA clustered damage sites. A class of non-DSB clustered DNA damage, corresponding to approximately 30% of the initial yield of DSBs, is converted into DSBs reflecting an artefact of preparation of genomic DNA for pulsed field gel electrophoresis. These clusters are removed within 4 min in both NHEJ-deficient and wild-type CHO cells. In xrs-5 cells, a proportion of non-DSB clustered DNA damage, representing approximately 10% of the total yield of non-DSB clustered DNA damage sites, are also converted into DSBs within approximately 30 min post-gamma but not post-alpha irradiation through cellular processing at 37 degrees C. That the majority of radiation-induced non-DSB clustered DNA damage sites are resistant to conversion into DSBs may be biologically significant at environmental levels of radiation exposure, as a non-DSB clustered damage site rather than a DSB, which only constitutes a minor proportion, is more likely to be induced in irradiated cells