Dietary resveratrol administration increases MnSOD expression and activity in mouse brain

trans-Resveratrol (3,4′,5-trihydroxystilbene; RES) is of interest for its reported protective effects in a variety of pathologies, including neurodegeneration. Many of these protective properties have been attributed to the ability of RES to reduce oxidative stress. In vitro studies have shown an increase in antioxidant enzyme activities following exposure to RES, including upregulation of mitochondrial superoxide dismutase, an enzyme that is capable of reducing both oxidative stress and cell death. We sought to determine if a similar increase in endogenous antioxidant enzymes is observed with RES treatment in vivo. Three separate modes of RES delivery were utilized; in a standard diet, a high fat diet and through a subcutaneous osmotic minipump. RES given in a high fat diet proved to be effective in elevating antioxidant capacity in brain resulting in an increase in both MnSOD protein level (140%) and activity (75%). The increase in MnSOD was not due to a substantial proliferation of mitochondria, as RES treatment induced a 10% increase in mitochondrial abundance (Citrate Synthase activity). The potential neuroprotective properties of MnSOD have been well established, and we demonstrate that a dietary delivery of RES is able to increase the expression and activity of this enzyme in vivo.     

Resveratrol interacts with estrogen receptor-β to inhibit cell replicative growth and enhance stress resistance by upregulating mitochondrial superoxide dismutase

trans-Resveratrol (RES) is one of a number of dietary polyphenols that have been reported to beneficially affect human physiology. Although numerous studies have attributed this to direct interactions between RES and histone deacetylases, recently the reliability of these results has been questioned. We have shown that the mitochondrial superoxide dismutase (MnSOD) is substantially upregulated in RES-treated cells. Here we explore the mechanisms underlying this, showing that two of RES's more interesting effects, inhibition of replication and enhancement of stress resistance, are mediated by MnSOD upregulation in three cell lines: MRC5 human lung fibroblasts, C2C12 mouse myoblasts, and SHSY5Y human neuroblastoma cells. When small interfering RNA was used to prevent induction of MnSOD expression, the effects of RES on population doubling time of cells in culture, and resistance to cell death after exposure to hydrogen peroxide or paraquat, were abolished. Interestingly, the RES-induced upregulation of MnSOD levels could be prevented by the estrogen receptor antagonist ICI 182780. RES's effects also could be reproduced using estradiol or the estrogen receptor-β agonist diarylpropionitrile, but not using the estrogen receptor-α agonist propylpyrazole triol. Thus, we suggest that RES interacts with estrogen receptor-β to induce the upregulation of MnSOD, which affects cell cycle progression and stress resistance. These results have important implications for our understanding of RES's biological activities and potential applications to human health.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Sensitization of rad mutants of Dictyostelium discoideum to ultraviolet light by postirradiation treatment with caffeine

The capacity of normal human cells to regulate DNA-repair pathways was examined. Synchronous populations of WI-38 human diploid fibroblasts were used to determine whether base-excision repair was increased as a function of the cell cycle. 2 parameters of the base-excision repair pathway were examined: (1) The induction of the DNA-repair enzyme uracil DNA glycosylase which functions in an initial step in base excision repair: (2) cell-mediated base-excision repair as measured by unscheduled DNA synthesis after exposure to sodium bisulfite or to methyl methanesulfonate. The glycosylase activity was increased 5-fold during cell proliferation; unscheduled DNA synthesis was enhanced 4- to 30-fold in a similar fashion. Equivalent results were observed where repair replication was quantitated using density-gradient analysis in the absence of hydroxyurea. The increase of the activity of the uracil DNA glycosylase and the enhancement of DNA repair occurred prior to the induction of DNA replication. Furthermore, at the maximal stimulation of DNA replication both glycosylase activity and DNA repair had substantially diminished. As the cells entered the second cell cycle, the glycosylase activity was again increased and then was again diminished. These results suggest that human cells actively modulate this DNA-repair pathway. The temporal stimulation of base-excision repair suggests the possibility that a DNA-repair complex may be formed prior to DNA replication to prescreen DNA and thus ensure the transfer of the correct genetic information to daughter cells.     

The combi-targeting concept: mechanism of action of the pleiotropic combi-molecule RB24 and discovery of a novel cell signaling-based combination principle

RB24 (NSC 741279), a 3-methyltriazene termed “combi-molecule” designed to possess mixed epidermal growth factor receptor (EGFR) targeting and DNA methylating properties showed over a 100-fold greater antiproliferative activity than Temodal^® (TEM), a 4-fold greater potency than gefitinib and a 5-fold stronger activity than an equi-effective combination of gefitinib + TEM against the O⁶-alkylguanine transferase (AGT)-proficient DU145 cell line that co-expresses EGFR. Investigation of the mechanisms underlying the unique potency of RB24 revealed that cell exposure to TEM was accompanied by activation of p38MAPK and concomitant elevation of the levels of X-ray repair cross-complementing group 1 (XRCC1) protein. Levels of phospho-p38MAPK and XRCC1 were increased by 2-fold in EGF-stimulated cells. In contrast, EGF-stimulation did not alter the status of these proteins in RB24-treated cells and this translated into a 2-fold lower level of XRCC1 when compared with those exposed to TEM + EGF. These effects correlated with significantly delayed DNA repair activity in combi-molecule-treated cells when compared with TEM-exposed ones. Further analysis demonstrated that in contrast to TEM, RB24 could block Bad phosphorylation at serine 136 in a dose-dependent manner and induced significantly higher levels of apoptosis than the former molecule. Tandem depletion of XRCC1 and Bad activation through alternative pathways using the MEK1 inhibitor, PD98059, led to substantial levels of apoptosis in RB24-treated cells. The results in toto indicate that the superior activity of the combi-molecule may be attributed to its ability to down-regulate DNA repair proteins such as XRCC1 and to alleviate anti-apoptotic signaling through blockade of EGFR-mediated signaling while inflicting high levels of DNA lesions to the cells.     

Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (K_d = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.     

Nitric oxide upregulates expression of DNA-PKcs to protect cells from DNA-damaging anti-tumour agents

Nitric-oxide synthase (NOS) activity has been detected in many human tumours, although its function is unclear. Here we show that exposure of cells to nitric oxide (NO) results in a 4-5-fold increase in expression of the DNA-dependent protein-kinase catalytic subunit (DNA-PKcs), one of the key enzymes involved in repairing double-stranded DNA breaks. This NO-mediated increase in enzymatically active DNA-PK not only protects cells from the toxic effects of NO, but also provides crossprotection against clinically important DNA-damaging agents, such as X-ray radiation, adriamycin, bleomycin and cisplatin. The NO-mediated increase in DNA-PKcs described here demonstrates the presence of a new and highly effective NO-mediated mechanism for DNA repair.     

SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD

To date no models exist to study MnSOD deficiency in human cells. To address this deficiency, we created a SOD2-null human cell line that is completely devoid of detectable MnSOD protein expression and enzyme activity. We utilized the CRISPR/Cas9 system to generate biallelic SOD2 disruption in HEK293T cells. These SOD2-null cells exhibit impaired clonogenic activity, which was rescued by either treatment with GC4419, a pharmacological small-molecule mimic of SOD, or growth in hypoxia. The phenotype of these cells is primarily characterized by impaired mitochondrial bioenergetics. The SOD2-null cells displayed perturbations in their mitochondrial ultrastructure and preferred glycolysis as opposed to oxidative phosphorylation to generate ATP. The activities of mitochondrial complex I and II were both significantly impaired by the absence of MnSOD activity, presumably from disruption of the Fe/S centers in NADH dehydrogenase and succinate dehydrogenase subunit B by the aberrant redox state in the mitochondrial matrix of SOD2-null cells. By creating this model we provide a novel tool with which to study the consequences of lack of MnSOD activity in human cells.     

Characterization of a UV-resistant strain,UVr-10, established from a human clonal cell line,RSb, with high sensitivity to UV, 4NQO,MNNG and interferon

Characterization was performed of a UV-resistant variant strain, UV^r-10, derived from a human clonal cell line, RSb, with high sensitivity not only to the lethal effect of 254-nm far-ultraviolet (UV) irradiation but also to the effects of 4-nitroquinoline 1-oxide (4NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and to the cell proliferation inhibition (CPI) effect of human leukocyte interferon (HuIFN-α) preparations.Colony-formation assays confirmed the increased resistance of UV^r-10 cells to both UV and 4NQO, but no increased resistance to MNNG. The marked recovery from the inhibition of the total cellular DNA synthesis of UV^r-10 cells, estimated by [methyl-³H]thymidine ([³H]dThd) uptake into the cellular DNA materials, was seen during 6 h after irradiation or 4NQO treatment even under the conditions without the recovery uptake into those of the parent RSb cells, but not during 6 h after MNNG treatment. Comparative studies on the activity of DNA repair synthesis between UV^r-10 and RSb cells, by measuring the extent of UV-, 4NQO- or MNNG-induced unscheduled DNA synthesis (UDS) and DNA repair replication, revealed an increased activity of UV^r-10 cells to UV and 4NQO but no significant increase of the activity to MNNG. These results suggest that increased DNA repair activities of a UV^r-10 cell line may account for its becoming resistant to the lethal effect of UV and 4NQO.Concerning the CPI effect of HuIFN-α, UV^r-10 cells showed increased resistance. Further, the DNA synthesis activity of UV^r-10 cells was not so inhibited by HuIFN-α exposure as that of RSb cells. However, HuIFN-α-exposed UV^r-10 cells showed more enhanced levels of activity of pppA(2′p5′A)_n synthetase (2–5A synthetase) than the exposed RSb, thus suggesting that HuIFN-α could exert enough intracellular effect even in UV^r-10 cells.The implication of the increased resistance of UV^r-10 cells to the effects of UV, 4NQO and HuIFN-α, but not to those of MNNG, is discussed.     

Cryopreservation and long-term storage of primary rat hepatocytes: Effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis

The effects of cryopreservation and long-term storage on substrate-specific cytochrome P45O-dependent activities and unscheduled DNA synthesis were studied in freshly isolated and cryopreserved hepatocytes derived from adult male Fischer 344 and Sprague-Dawley rats. Primary rat hepatocytes were isolated via an in situ collagenase perfusion technique, cryopreserved at –196°C, and thawed at 5 weeks and 104 and 156 weeks post-freezing. In Fischer 344 and Sprague-Dawley rats, cryopreserved hepatocytes were equivalent or similar to freshly isolated hepatocytes in substrate-specific activities for 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase and unscheduled DNA synthesis responses. No significant differences in activities toward 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase, the substrate-specific activities for cytochromes P4501A1 and P4501A2 and cytochrome P4502E1, respectively, were observed between freshly isolated and cryopreserved hepatocytes. Similar unscheduled DNA synthesis responses, a measure of DNA damage and repair, were observed after exposure to the genotoxic carcinogens 2-acetylaminofluorene, 7,12-dimethyEbenz[a]anthracene, and dimethylnitrosamine; although some decreases were also observed in Fischer 344 hepatocytes after 104 weeks and Sprague-Dawley hepatocytes after 156 weeks in the highest concentrations tested. These results suggest that cryopreserved hepatocytes, stored for extended periods of time in liquid nitrogen, are metabolically equivalent to freshly isolated hepatocytes in their ability to activate precarcinogens.Abbreviations 2-AAF 2-acetylaminofluorene - DDH₂O distilled deionized water - DMBA 7,12-dimethyIbenz[a]anthracene - DMN dimethylnitrosamine - DMNA dimethylnitrosamine-N-demethylase - DMSO dimethyl sulfoxide - EROD 7-ethoxyresorufin-O-deethylase - F344 Fischer 344 - FBS fetal bovine serum - %IR percentage of cells in repair - LN₂ liquid nitrogen - LSD least significant difference - CG cytoplasmic grains - NNG net nuclear grains - SD Sprague-Dawley - UDS unscheduled DNA synthesis - WE Williams' Medium E     

Evaluating long-term cellular effects of the arsenic species thio-DMAV: qPCR-based gene expression as screening tool

Thio-dimethylarsinic acid (thio-DMA^V) is a human urinary metabolite of the class 1 human carcinogen inorganic arsenic as well as of arsenosugars. Thio-DMA^V exerts strong cellular toxicity, whereas its toxic modes of action are not fully understood. For the first time, this study characterises the impact of a long-term (21 days) in vitro incubation of thio-DMA^V on the expression of selected genes related to cell death, stress response, epigenetics and DNA repair. The observed upregulation of DNMT1 might be a cellular compensation to counterregulate the in a very recent study observed massive global DNA hypomethylation after chronic thio-DMA^V incubation. Moreover, our data suggest that chronic exposure towards subcytotoxic, pico- to nanomolar concentrations of thio-DMA^V causes a stress response in human urothelial cells. The upregulation of genes encoding for proteins of DNA repair (Apex1, Lig1, XRCC1, DDB2, XPG, ATR) as well as damage response (GADD45A, GADD45G, Trp53) indicate a potential genotoxic risk emanating from thio-DMA^V after long-term incubation.     

Analysis of metal-induced DNA lesions and DNA-repair replication in mammalian cells

The potency of several metal compounds in causing lesions in DNA either directly or by exposure of intact cultured cells has been examined using the neutral conditions of nucleoid gradient sedimentation. HgCl2 was clearly the most potent inducer of single-strand breakage when added to isolated nucleoids or when nucleoids were prepared from cells treated with this compound. CaCrO4 , however, caused DNA-strand breaks in nucleoids isolated from cells treated with this agent but did not induce DNA strand breaks when added directly to nucleoids. Although less potent than HgCl2, NiCl2 also caused significant single strand breakage in isolated nucleoids or in nucleoids prepared from cells treated with this metal. Since strand breakage of DNA in intact cells may occur secondary to activation of DNA-dependent nucleases during repair replication, CsCl gradient density sedimentation was utilized to examine whether repair processes were induced by exposure of cells to NiCl2, HgCl2 and CaCrO4 . CaCrO4 and NiCl2 induced substantial DNA-repair activity at concentrations and exposure times where DNA lesions could not be detected whereas HgCl2 induced a 10-fold lower level of DNA-repair activity compared to CaCrO4 at optimal concentrations which again were below the concentrations of this metal that produced measurable DNA lesions. Both the induction of DNA-repair activity and DNA-strand breakage by these metals was concentration- and time-dependent. These results demonstrate some unique aspects of the interaction of HgCl2, NiCl2 and CaCrO4 with the DNA of intact cells and point to the possible important correlation of induction of DNA repair to carcinogenesis since nickel and chromate have clearly been implicated as carcinogens and induce considerable repair whereas HgCl2 is not considered a carcinogen and induces the least DNA repair despite its potency in producing DNA lesions.     

Methylation damage to RNA induced in vivo in Escherichia coli is repaired by endogenous AlkB as part of the adaptive response

Cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions induced in DNA and RNA in vitro and in pre-damaged DNA and RNA bacteriophages in vivo are repaired by the Escherichia coli (E. coli) protein AlkB and a human homolog, ALKBH3. However, it is not known whether endogenous RNA is repaired in vivo by repair proteins present at physiological concentrations. The concept of RNA repair as a biologically relevant process has therefore remained elusive. Here, we demonstrate AlkB-mediated repair of endogenous RNA in vivo by measuring differences in lesion-accumulation in two independent AlkB-proficient and deficient E. coli strains during exposure to methyl methanesulfonate (MMS). Repair was observed both in AlkB-overproducing strains and in the wild-type strains after AlkB induction. RNA repair appeared to be highest in RNA species below 200 nucleotides in size, mainly comprising tRNAs. Strikingly, at least 10-fold more lesions were repaired in RNA than in DNA. This may be a consequence of some 30-fold higher levels of aberrant methylation in RNA than in DNA after exposure to MMS. A high primary kinetic isotope effect (>10) was measured using a deuterated methylated RNA substrate, D₃-1me(rA), demonstrating that it is the catalytic step, and not the search step that is rate-limiting. Our results demonstrate that RNA repair by AlkB takes place in endogenous RNA as part of an adaptive response in wild-type E. coli cells.     

Dual effects of α-synuclein on neurotoxicity induced by low dosage of rotenone are dependent on exposure time in dopaminergic neuroblastoma cells

This study was designed to investigate the effects of α-synuclein on toxicity induced by long-term exposure to relatively low concentrations of rotenone.Compared with the control groups,the inhibition of cell viability which overexpressed α-synuclein(SH-SY5Y-Syn) improved after 1 and 2 weeks of rotenone treatment.The complex I activity was greater and the mitochondrial membrane swelling intensity was reduced after 1 and 2 weeks of treatment,which indicated that α-synuclein,at least in part,resists the rotenone-induced oxidative stress.The results indicate that α-synuclein has a dual effect on toxicity of rotenone according to exposure time in human SH-SY5Y cells.     

Molecular manifestations of radiation-induced genome instability: the possibility of chemical modification

The molecular manifestations of radiation-induced genome instability-changes of the DNA structure, the excision DNA repair and the contents of the reactive oxygen forms in bone marrow cells of the repair proficient mice (CBA) and of the repair-defective (101/H) lines in the dynamics up to 185 day after ionizing radiation exposure in the dose of 1.5 Gy were studied. Is was established, that after irradiation in bone marrow cells the descendants with the decreased activity of excision DNA repair and prone to increased changes of DNA structure DHK is arised. The injection of the phenozane in concentrations causing its receptor interaction with cells, did not defend DNA of the bone marrow cells from the radiation injury after the exposure in a sublethal dose, however it exerted influence on long-term changes. Due to the phenosane of the bone marrow cells of the irradiated mice of CBA line exhibited the larger activity in a DNA repair from damages and maintenance of vitality. The bone marrow cells of male mice of repair defective 101/H line, which phenozan was entered before the irradiation, remained unfit to the remuval of DNA damages by the repair, that probably resulted the activations of the program of the maintenance of genome constancy by the apoptosis in the cells--carriers of the structural defects and the cause of animal lethality.     

白藜芦醇对乳腺癌细胞MCF-7增殖的影响

目的：研究白藜芦醇体外活性,确定它的植物雌激素作用。方法：采用MTT法观察不同浓度白藜芦醇对MCF-7细胞增殖作用的影响。采用DNA ladder法和荧光显微镜观察高浓度白藜芦醇对细胞的影响。免疫组化法观察低浓度白藜芦醇对核增殖抗原PCNA表达的影响。结果：MTT结果显示白藜芦醇高浓度抑制MCF-7细胞增殖,IC50为8.70×10-5mol/L;低浓度（10-7~10-6mol/L）则对细胞有促增殖作用,最高促增殖浓度为1.0×10-7mol/L。DNA ladder和荧光显微镜可观察到高浓度白藜芦醇作用后细胞典型的凋亡形态。免疫组化结果显示低浓度白藜芦醇作用后,细胞核内PCNA表达明显增加（P〈0.05）。结论：高、低浓度的白藜芦醇对MCF-7细胞分别表现为诱导凋亡和促增殖作用,呈现出植物雌激素对MCF-7细胞典型的双向调节作用。     

Effects of transgene expression of superoxide dismutase and glutathione peroxidase on pulmonary epithelial cell growth in hyperoxia

Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50-300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O(2) for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells (P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD (P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury.     

New insights in the removal of the hydantoins, oxidation product of pyrimidines, via the base excision and nucleotide incision repair pathways

Background

Oxidative damage to DNA, if not repaired, can be both miscoding and blocking. These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER) and nucleotide incision repair (NIR). Hydantoin derivatives such as 5-hydroxyhydantoin (5OH-Hyd) and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd), major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Hydantoins are blocking lesions for DNA polymerases and excised by bacterial and yeast DNA glycosylases in the BER pathway. However little is known about repair of pyrimidine-derived hydantoins in human cells.

Methodology/Principal Findings

Here, using both denaturing PAGE and MALDI-TOF MS analyses we report that the bacterial, yeast and human AP endonucleases can incise duplex DNA 5′ next to 5OH-Hyd and 5OH-5Me-Hyd thus initiating the NIR pathway. We have fully reconstituted the NIR pathway for these lesions in vitro using purified human proteins. Depletion of Nfo in E. coli and APE1 in HeLa cells abolishes the NIR activity in cell-free extracts. Importantly, a number of redundant DNA glycosylase activities can excise hydantoin residues, including human NTH1, NEIL1 and NEIL2 and the former protein being a major DNA glycosylase activity in HeLa cells extracts.

Conclusions/Significance

This study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions in vivo.     

Cadmium inhibits human DNA mismatch repair in vivo

The heavy metal cadmium (Cd) is a human carcinogen that inhibits DNA repair activities. We show that DNA mismatch repair (MMR)-mediated cell cycle arrest after alkylation damage is suppressed by exposure to Cd and that this effect is reversed by preincubation with excess of zinc (Zn). We show that Cd-mediated inactivation of MMR activity is not caused by disruption of complex formation between the MMR proteins hEXO1-hMutS alpha and hEXO1-hMutL alpha nor does Cd inhibit 5'-exonuclease activity of hEXO1 in vitro. Thus, our studies show that exposure of human cells to Cd suppresses MMR activity, a repair activity known to play an important role in colon cancer and that this effect can be reversed by Zn treatment.