title: 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 use 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 problem, we introduced into E. coli the Drosophila deoxynucleoside kinase (Dm-dNK), 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 properties 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.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48202, United States.
authors.forename: Brittany
authors.initials: B
authors.lastname: Betham
authors.forename: Sophia
authors.initials: S
authors.lastname: Shalhout
authors.forename: Victor E
authors.initials: VE
authors.lastname: Marquez
authors.forename: Ashok S
authors.initials: AS
authors.lastname: Bhagwat
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001374:Azacitidine / Q000737:chemistry / Q000633:toxicity; D003562:Cytidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000633:toxicity; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D053843:DNA Mismatch Repair / Q000187:drug effects; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000633:toxicity*; D004331:Drosophila melanogaster / Q000187:drug effects / Q000201:enzymology*; D004926:Escherichia coli / Q000187:drug effects*; D005453:Fluorescence; D008826:Microbial Sensitivity Tests; D016296:Mutagenesis / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D003841:Deoxycytidine; D003562:Cytidine; C009131:pyrimidin-2-one beta-ribofuranoside; D004247:DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D001374:Azacitidine