Homology modeling of NAD+-dependent DNA ligase of the Wolbachia endosymbiont of Brugia malayi and its drug target potential using dispiro-cycloalkanones.

Lymphatic filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility, and viability and thus has great promise as a novel approach for treating filarial diseases. NAD(+)-dependent DNA ligase is an essential enzyme of DNA replication, repair, and recombination. Therefore, in the present study, the antifilarial drug target potential of the NAD(+)-dependent DNA ligase of the Wolbachia symbiont of Brugia malayi (wBm-LigA) was investigated using dispiro-cycloalkanone compounds. Dispiro-cycloalkanone specifically inhibited the nick-closing and cohesive-end ligation activities of the enzyme without inhibiting human or T4 DNA ligase. The mode of inhibition was competitive with the NAD(+) cofactor. Docking studies also revealed the interaction of these compounds with the active site of the target enzyme. The adverse effects of these inhibitors were observed on adult and microfilarial stages of B. malayi in vitro, and the most active compounds were further monitored in vivo in jirds and mastomys rodent models. Compounds 1, 2, and 5 had severe adverse effects in vitro on the motility of both adult worms and microfilariae at low concentrations. Compound 2 was the best inhibitor, with the lowest 50% inhibitory concentration (IC50) (1.02 µM), followed by compound 5 (IC50, 2.3 µM) and compound 1 (IC50, 2.9 µM). These compounds also exhibited the same adverse effect on adult worms and microfilariae in vivo (P < 0.05). These compounds also tremendously reduced the wolbachial load, as evident by quantitative real-time PCR (P < 0.05). wBm-LigA thus shows great promise as an antifilarial drug target, and dispiro-cycloalkanone compounds show great promise as antifilarial lead candidates.

Arsenicals affect base excision repair by several mechanisms.

Inorganic arsenic is a strong, widespread human carcinogen. How exactly inorganic arsenic exerts carcinogenicity in humans is as yet unclear, but it is thought to be closely related to its metabolism. At exposure-relevant concentrations arsenic is neither directly DNA reactive nor mutagenic. Thus, more likely epigenetic and indirect genotoxic effects, among others a modulation of the cellular DNA damage response and DNA repair, are important molecular mechanisms contributing to its carcinogenicity. In the present study, we investigated the impact of arsenic on several base excision repair (BER) key players in cultured human lung cells. For the first time gene expression, protein level and in case of human 8-oxoguanine DNA glycosylase 1 (hOGG1) protein function was examined in one study, comparing inorganic arsenite and its trivalent and pentavalent mono- and dimethylated metabolites, also taking into account their cellular bioavailability. Our data clearly show that arsenite and its metabolites can affect several cellular endpoints related to DNA repair. Thus, cellular OGG activity was most sensitively affected by dimethylarsinic acid (DMA(V)), DNA ligase IIIα (LIGIIIα) protein level by arsenite and X-ray cross complementing protein 1 (XRCC1 protein) content by monomethylarsonic acid (MMA(V)), with significant effects starting at ≥3.2µM cellular arsenic. With respect to MMA(V), to our knowledge these effects are the most sensitive endpoints, related to DNA damage response, that have been identified so far. In contrast to earlier nucleotide excision repair related studies, the trivalent methylated metabolites exerted strong effects on the investigated BER key players only at cytotoxic concentrations. In summary, our data point out that after mixed arsenic species exposure, a realistic scenario after oral inorganic arsenic intake in humans, DNA repair might be affected by different mechanisms and therefore very effectively, which might facilitate the carcinogenic process of inorganic arsenic.

Stimulation of DNA repair in Saccharomyces cerevisiae by Ginkgo biloba leaf extract.

Many extracts prepared from plants traditionally used for medicinal applications contain a variety of phytochemicals with antioxidant and antigenotoxic activity. In this work we measured the DNA protective effect of extracts of Ginkgo biloba leaves from oxidative stress using Saccharomyces cerevisiae as experimental model. The extract improved viability of yeast cells under oxidative stress imposed by hydrogen peroxide. In accordance with previous reports on antioxidant properties of G. biloba extracts, pre-incubation of yeast cells promoted a decrease in intracellular oxidation. We assessed DNA damage by our recently developed yeast comet assay protocol. Upon oxidative shock, DNA damage decreased in a dose-dependent manner in experiments of pre-incubation and simultaneous incubation with the extract, indicating a direct protective effect. In addition, the extract improved DNA repair rate following oxidative shock as measured by faster disappearance of comet tails. This suggests that the extract stimulates the DNA repair machinery in its DNA protective action in addition to directly protect DNA from oxidation. The observed DNA repair depends on the DNA repair machinery since no DNA repair was observed under restrictive conditions in a conditional mutant of the CDC9 gene (Accession No. Z74212), encoding the DNA ligase involved in the final step of both nucleotide and base excision repair.

Extremolyte-like applicability of an archaeal exopolymer, poly-gamma-L-glutamate.

An extremely halophilic archaeon Natrialba aegyptiaca produces extracellular poly-gamma-glutamate (PGA), in which only L-glutamate is polymerized via gamma-amide linkages. We examined the extremolyte-like applicability of archaeal PGA and found the ameliorating effects of L-PGA on the resistibility to freeze-thawing and proteolysis, thermostability, and alkalotolerance of a model enzyme, labile DNA ligase. For example, the coexistence of low (e.g. 0.01 mg mL(-1)) and high (e.g. 0.1 mg mL(-1)) concentrations of L-PGA with an average molecular mass of 1000 kDa increased the midpoint of thermal inactivation of DNA ligase by about 15 degrees C and 18 degrees C, respectively, and the model enzyme further remained active even under extremely alkaline conditions of pH 11.4 in the presence of the high concentration of L-PGA. This is the first characterization of the stereo-regular PGA molecules as atypical extremolytes. L-PGA from extremophiles has great potential as a bio-based protectant (or stabilizer) with industrial versatility.

CsA can induce DNA double-strand breaks: implications for BMT regimens particularly for individuals with defective DNA repair.

Several human disorders mutated in core components of the major DNA double-strand break (DSB) repair pathway, non-homologous end joining (NHEJ), have been described. Cell lines from these patients are characterized by sensitivity to DSB-inducing agents. DNA ligase IV syndrome (LIG4) patients specifically, for unknown reasons, respond particularly badly following treatment for malignancy or BMT. We report the first systematic evaluation of the response of LIG4 syndrome to compounds routinely employed for BMT conditioning. We found human pre-B lymphocytes, a key target population for BMT conditioning, when deficient for DNA ligase IV, unexpectedly exhibit significant sensitivity to CsA the principal prophylaxis for GVHD. Furthermore, we found that CsA treatment alone or in combination with BU and fludarabine resulted in increased levels of DSBs specifically in LIG4 syndrome cells compared to wild-type or Artemis-deficient cells. Our study shows that CsA can induce DSBs and that LIG4 syndrome patient's fail to adequately repair this damage. These DSBs likely arise as a consequence of DNA replication in the presence of CsA. This work has implications for BMT and GVHD management in general and specifically for LIG4 syndrome.

DNA ligase IV suppresses medulloblastoma formation.

Substantial neural defects are often present in mice with targeted inactivation of DNA repair factors such as DNA ligase IV (Lig4). Whereas Lig4(-/-) mice undergo widespread neural apoptosis and die during development, p53 deficiency rescues this death. We found that all Lig4(-/-)p53(-/-) mice developed medulloblastoma, but did not develop other tumors of the nervous system. Lig4(-/-)p53(-/-) medulloblastoma occurred as early as 21 days of age, originated in the external granule layer of the developing cerebellum, and was synaptophysin immunoreactive. These data reveal a pronounced susceptibility of the cerebellum to the effects of chronic DNA damage and provide a direct link between genotoxic stress and medulloblastoma formation.

Treatment of human cells with N-Nitroso(acetoxymethyl)methylamine: distribution patterns of piperidine-sensitive DNA damage at the nucleotide level of resolution are related to the sequence context.

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) present in tobacco smoke is a major carcinogen involved in tobacco-induced lung cancer. Its complex bioactivation along two pathways, which leads to methylation and pyridyloxobutylation of DNA, makes the study of NNK-induced DNA damage difficult. We selected two nitroso compounds, N-methyl-N-nitrosourea (MNU) and N-nitroso(acetoxymethyl)methylamine (NDMAOAc), with which to map NNK-induced DNA methylation frequency at every nucleotide position. We address the issue of how sequence context and complex chromatin structures, present in living cells, regulate the formation of modified purines through methylation generated by MNU and NDMAOAc. For comparison purposes, purified DNA was treated with dimethyl sulfate (DMS). We used ligation-mediated polymerase chain reaction to map and conduct a high-resolution footprinting analysis of the DNA damage along the p53 gene (exons 5-8), the ras gene family (exons 1 and 2 of H-, K-, and N-ras genes), and the c-jun promoter in living cells. The distribution of piperidine-sensitive DNA damage induced in cellular DNA and purified DNA by MNU or NDMAOAc was identical. MNU and NDMAOAc methylate more frequently the central guanines in a run of guanines, suggesting a regioselective mechanism for DNA methylation. In contrast, DMS methylates more frequently guanines at the 5'-end of a guanine run; this frequency decreased from the 5'- to the 3'-end. While the presence of adenines in a guanine run does not affect the distribution pattern, the presence of pyrimidines does change said pattern. Our data lead us to suggest that NNK would also methylate DNA sequences in a way similar to that of MNU or NDMAOAc. Footprinted areas of DNA methylated with MNU or NDMAOAc correspond to a consensus sequence for transcription factors AP-1, NF-Jun, CCAAT box, SP-1, and RSRF, as observed in c-jun promoters. Our results are in line with the fact that NNK metabolites, generated through the alpha-hydroxylation pathways, could potentially be mutagenic, since these activated metabolites can methylate guanines. In p53 and ras genes, the frequency of methylation of guanines parallels the frequency of mutations of those same guanines in lung cancer.

Signalling via receptor tyrosine kinase modulates the expression of the DNA repair enzyme XPD in cultured cells.

Oxidative damage to DNA has been documented in cells isolated from subjects with diabetes. Herein, we evaluate the mechanism(s) that regulate the expression of the DNA repair enzyme XPD. CHO cells transfected with the human insulin receptor (CHO/HIRc) showed a threefold increase in the level of XPD mRNA when compared to control CHO/neo cells (P < 0.01). The addition of insulin to serum-starved cells led to an increase in XPD mRNA levels in both CHO/neo and CHO/HIRc cells, in a time and dose dependent fashion. Insulin acted primarily by inducing XPD transcription. Moreover, inhibition of protein synthesis by cyclohexamide induced a marked degradation of XPD mRNA levels in insulin treated cells. Site-directed mutagenesis of the tyrosine-kinase domain of the insulin receptor abolished the increase in XPD mRNA resulting from the transfection with wild type insulin receptors (P < 0.001). Western blot analysis of cell extracts from CHO/neo and CHO/HIRc cells revealed an increase in XPD counterpart protein was also induced by transfecting cells with the human insulin receptor. Evaluation of DNA damage by means of internucleosomal fragmentation showed a dramatic decrease in DNA fragmentation in CHO cells transfected with wild-type insulin receptor compared to control CHO/neo cells. DNA fragmentation was further decreased by the addition of insulin in the culture medium. In summary, our data indicates that activation of the insulin receptor plays an important role in the cellular response leading to repair of damaged DNA.

Modulation by dietary copper of aflatoxin B1-induced activity of DNA repair enzymes poly (ADP-ribose) polymerase, DNA polymerase beta and DNA ligase.

The activity of some nuclear enzymes associated with DNA repair was examined following aflatoxin B1 administration in rats maintained on different levels of dietary copper. Induction of poly(ADP-ribose) polymerase, DNA polymerase beta and DNA ligase was found to be significantly higher in copper-deficient rats. Copper supplementation, even at marginal doses, was able to bring down the induction to the level observed in normal rats. The results emphasize the protective role of copper against the DNA damaging effects of aflatoxin B1.

Modulation of carcinogen-induced DNA damage and repair enzyme activity by dietary riboflavin.

Formation of single strand breaks in nuclear DNA induced by hepatocarcinogens aflatoxin B1 and N-nitrosodimethylamine was observed to be more pronounced in rats maintained on a riboflavin-deficient diet compared to that on a normal diet. This increased damage was reversed on riboflavin supplementation. The induction of repair enzymes poly(ADP-ribose) polymerase, DNA polymerase beta and DNA ligase was significantly higher in riboflavin-deficient rats following DNA damage caused by the administration of carcinogens. Riboflavin supplementation brought down the induction to the levels found in rats maintained on normal diet. Since damage to DNA and its altered repair may relate to carcinogenesis, modulation of these parameters by riboflavin suggests a potential chemopreventive role of this vitamin.

Glutathione can rescue the inhibitory effects of nickel on DNA ligation and repair synthesis.

The purpose of this investigation was to explore the reason why nickel chloride enhances the cytotoxicity and genotoxicity of ultraviolet (UV) light, but not that of methyl methanesulfonate (MMS) in Chinese hamster ovary cells. The cellular glutathione content was increased by treatment with MMS or nickel, but not with UV. Post-treatment with nickel synergistically raised the cellular glutathione content in MMS-treated cells; this phenomenon was not observed in UV-irradiated cells. Preventing cellular glutathione induction by buthionine sulfoximine increased the cytotoxicity, the frequency of sister chromatid exchange and prolonged the cell cycle in cells treated with nickel or MMS plus nickel. Pretreatment with N-acetylcysteine, a glutathione precursor, increased the clonogenic survival of cells treated with UV plus nickel. In vitro assays indicated that nickel could inhibit oligonucleotide ligation and the repair synthesis of UV- or MMS-treated plasmids and glutathione could relieve nickel inhibition. These results suggest that the enhancement by nickel of UV cytotoxicity and genotoxicity may be due to its inhibition of DNA repair, whereas treating cells with MMS plus nickel increased cellular glutathione levels, which may help in neutralizing the toxicity of nickel. The results also suggest that the activity of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, may be increased by treatment with MMS, nickel and more so with MMS plus nickel.

Synthesis and biochemical evaluation of the CBI-PDE-I-dimer, a benzannelated analog of (+)-CC-1065 that also produces delayed toxicity in mice.

A practical synthesis of CBI (2) was developed and applied to the synthesis of benzannelated analogs of CC-1065, including CBI-PDE-I-dimer (13) and CBI-bis-indole [(+)-A'BC]. The CBI-PDE-I-dimer was shown to have similar DNA sequence selectivity and structural effects on DNA as (+)-CC-1065. Of particular importance was the observed duplex winding effect that has been associated with the pyrrolidine ring of the nonalkylated subunits of (+)-CC-1065 and possibly correlated with its delayed toxicity effects. The effect of CBI-PDE-I-dimer was also compared to (+)-CC-1065 in the inhibition of duplex unwinding by helicase II and nick sealing by T4 ligase and found to be quantitatively similar. The in vitro and in vivo potencies of the CBI compounds corresponded very closely to the corresponding CPI derivatives. Finally, CBI-PDE-I-dimer was like (+)-CC-1065 in causing delayed toxicity in mice.