Use of Drosophila deoxynucleoside kinase to study mechanism of toxicity and mutagenicity of deoxycytidine analogs in Escherichia coli

Abstract

Most bacteria, including Escherichia coli, lack an enzyme that can phosphorylate deoxycytidine and its analogs. Consequently, most studies of toxicity and mutagenicity of cytosine analogs u se ribonucleosides such as 5-azacytidine (AzaC) and zebularine (Zeb) instead of their deoxynucleoside forms, 5-aza-2'-deoxycytidine (AzadC) and 2'-deoxy-zebularine (dZeb). The former analogs are incorporated into both RNA and DNA creating complex physiological responses in cells. To circumvent this p roblem, we introduced into E. coli the Drosophila deoxynucleoside kinase (DmdNK), which has a relaxed substrate specificity, and tested these cells for sensitivity to AzadC and dZeb. We find that Dm-dNK expression increases substantially sensitivity of cells to these analogs and dZeb is very mutagenic in cells expressing the kinase. Furthermore, toxicity of dZeb in these cells requires DNA mismatch correction system suggesting a mechanism for its toxicity and mutagenicity. The fluorescence p roperties of dZeb were used to quantify the amount of this analog incorporated into cellular DNA of mismatch repair-deficient cells expressing Dm-dNK and the results showed that in a mismatch correction-defective strain a high percentage of DNA bases may be replaced with the analog without long term toxic effects. This study demonstrates that the mechanism by which Zeb and dZeb cause cell death is fundamentally different than the mechanism of toxicity of AzaC and AzadC. It also opens up a new way to study the mechanism of action of deoxycytidine analogs that are used in anticancer chemotherapy.

Cytosine DNA methylation influences drug resistance inEscherichia colithrough increasedsugEexpression

“…This may be due to the fact that there is indeed a small repressive effect of DNA methylation on sugE expression at early logarithmic phase, and/or stationary phase cells that are not rapidly dividing are not as likely to incorporate as much 5‐azacytidine into DNA. In addition, 5‐azacytidine is known to be toxic to E. coli in killing assays (Bhagwat & Roberts, ; Betham et al ., ). In our experiments, there are lower A 600 nm readings only after c .…”

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis

“…This block is weaker than MMC cross-links, but stronger than DNA double-strand breaks induced by bleocin, with the latter proposed to be repaired in a cell cycle stage-independent manner (Schubert et al, 2004) To explore the detoxification mechanism of zebularine-induced DNA damage further, we analyzed the sensitivity of mutants of several DNA repair pathways. In bacteria, mutants defective in NER were hypersensitive to 5-azacytidine (Betham et al, 2010). Therefore, we exposed plants mutated in the XERODERMA PIG-MENTOSUM GROUP F (XPF) gene, the endonuclease involved in NER and removal of nonhomologous overhangs in intramolecular homologous recombination events (Gaillard and Wood, 2001; (A) Effects of zebularine-atr on gene transcript levels.…”

Adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase mutants combined with gemcitabine harbor a safe cancer treatment profile

“…The broad substrate specificity of this enzyme, along with its high catalytic rate, makes it unique among the known dNKs. Expression of Dm-dNK in cancer cells increases the sensitivity to several cytotoxic nucleoside analogs (2)(3)(4), rendering the enzyme a candidate for possible use as a suicide gene with combined gene therapy and chemotherapy.…”