WRN Exonuclease activity is blocked by specific oxidatively induced base lesions positioned in either DNA strand

Werner syndrome (WS) is a premature aging disorder caused by mutations in the WS gene (WRN). Although WRN has been suggested to play an important role in DNA metabolic pathways, such as recombination, replication and repair, its precise role still remains to be determined. WRN possesses ATPase, helicase and exonuclease activities. Previous studies have shown that the WRN exonuclease is inhibited in vitro by certain lesions induced by oxidative stress and positioned in the digested strand of the substrate. The presence of the 70/86 Ku heterodimer (Ku), participating in the repair of double-strand breaks (DSBs), alleviates WRN exonuclease blockage imposed by the oxidatively induced DNA lesions. The current study demonstrates that WRN exonuclease is inhibited by several additional oxidized bases, and that Ku stimulates the WRN exonuclease to bypass these lesions. Specific lesions present in the non-digested strand were shown also to inhibit the progression of the WRN exonuclease; however, Ku was not able to stimulate WRN exonuclease to bypass these lesions. Thus, this study considerably broadens the spectrum of lesions which block WRN exonuclease progression, shows a blocking effect of lesions in the non-digested strand, and supports a function for WRN and Ku in a DNA damage processing pathway

Effect of endotoxemia in suckling rats on pancreatic integrity and exocrine function in adults : a review report

Background. Endotoxin (LPS), the component of Gram-negative bacteria, is responsible for sepsis and neonatal mortality, but low concentrations of LPS produced tissue protection in experimental studies. The effects of LPS applied to the suckling rats on the pancreas of adult animals have not been previously explored. We present the impact of neonatal endotoxemia on the pancreatic exocrine function and on the acute pancreatitis which has been investigated in the adult animals. Endotoxemia was induced in suckling rats by intraperitoneal application of LPS from Escherichia coli or Salmonella typhi. In the adult rats, pretreated in the early period of life with LPS, histological manifestations of acute pancreatitis have been reduced. Pancreatic weight and plasma lipase activity were decreased, and SOD concentration was reversed and accompanied by a significant reduction of lipid peroxidation products (MDA + 4 HNE) in the pancreatic tissue. In the pancreatic acini, the significant increases in protein signals for toll-like receptor 4 and for heat shock protein 60 were found. Signal for the CCK1 receptor was reduced and pancreatic secretory responses to caerulein were diminished, whereas basal enzyme secretion was unaffected. These pioneer studies have shown that exposition of suckling rats to endotoxin has an impact on the pancreas in the adult organism

Oxidation products of 5-methyl cytosine are decreased in senescent cells and tissues of progeroid mice

5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1−/− primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2′-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2′-deoxyuridine and 5-(hydroxymethyl)-2′-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)- 2′-deoxycytidine and 5-formyl-2′-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2′-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence

A mild and efficient approach to the 6H-oxazolo[3,2-f]pyrimidine-5,7-dione scaffold via unexpected rearrangement of 2,3-dihydropyrimido[6,1-b][1,5,3]dioxazepine-7,9(5H,8H)-diones:synthesis, crystallographic studies, and cytotoxic activity screening

We report a mild and efficient approach to the optically pure 6H-oxazolo[3,2-f]pyrimidine-5,7-dione scaffold via the unexpected rearrangement and ring contraction of 2,3-dihydropyrimido[6,1-b][1,5,3]- dioxazepine-7,9(5H,8H)-diones derived from nucleoside precursors. The developed procedure enables the synthesis of a wide range of compounds with great structural diversity. The structure of the obtained compounds was confirmed by NMR spectroscopy and single crystal X-ray structural analysis. The final products were tested for cytotoxic effect on one non-cancerous (fibroblasts) and six cancer cell lines of different origins (colon, glioma, breast, cervix, vulvar, and lung). The synthesized products are low molecular weight compounds with lead-like properties suitable for a medicinal chemistry optimization program

ERCC1-deficient cells and mice are hypersensitive to lipid peroxidation

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1-/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1-/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1-/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats

Spontaneous DNA damage to the nuclear genome promotes senescence, T redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Spontaneous DNA damage to the nuclear genome promotes senescence,redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Different faces of DNA repair - Nobel price 2015 in chemistry

W 2015 r. Nagroda Nobla w dziedzinie chemii została przyznana za badania mechanistyczne nad naprawą DNA Paulowi Modrichowi, Tomasowi Lindahlowi i Azizowi Sancarowi. Paul Modrich pracuje w Howard Hughes Medical Institute oraz Duke University School of Medicine, Durham, USA. Nagrodą zostały wyróżnione jego prace nad naprawą źle dopasowanych zasad, które powstają głównie podczas replikacji, zaś ten typ naprawy jest "pierwszą linią ochrony" stabilności genomu. Tomas Lindahl jest profesorem chemii medycznej i fizycznej, emerytowanym dyrektorem Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Wielka Brytania. Nagrodę Nobla otrzymał za odkrycia w dziedzinie naprawy przez wycięcie zasady usuwającej z DNA niewielkie uszkodzenia, głównie oksydacyjne i alkilacyjne. Aziz Sancar jest profesorem biochemii i biofizyki na University of North Carolina School of Medicine, Chapel Hill, USA. Nagrodę Nobla otrzymał za osiągnięcia w dziedzinie naprawy przez wycięcie nukleotydu. System ten usuwa z DNA duże modyfikacje takie jak dimery pirymidynowe indukowane światłem ultrafioletowym. Badania uczonych stworzyły podwaliny pod zrozumienie mechanizmu ewolucji świata ożywionego, a także procesów nowotworowych i opracowanie nowoczesnych terapii.The Nobel Prize in chemistry for 2015 was awarded to Paul Modrich, Tomas Lindahl and Aziz Sancar for mechanistic studies on DNA repair. Paul Modrich works in Howard Hughes Medical Institute and Duke University School of Medicine, Durham, USA. The prize has been awarded for his work on Mismatch Repair, which removes mismatched nucleotides formed mainly during replication and is the "first line of defense" of genome stability. Tomas Lindahl is a professor of medical and physical chemistry, emeritus director of Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, United Kingdom. The Nobel Prize was awarded to him for discoveries on Base Excision Repair, which removes from the DNA small lesions, mainly alkylated and oxidatively formed damages. Aziz Sancar is a professor in biochemistry and biophysics at University of North Carolina School of Medicine, Chapel Hill, USA. He was awarded for the achievements on Nucleotide Excision Repair. The system removes from the DNA big lesions, such as pyrimidine dimers induced by ultraviolet light. Studies of these researchers made a basis for understanding of the evolution of living world as well as carcinogenic process and for elaboration of novel therapies