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Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors
M Takahashi1, M Nishizawa, K Negishi
1Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Abstract:
To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.
Insights
N4-aminocytidine induces mutations by incorporating into DNA as N4-aminodeoxycytidine 5'-triphosphate (dCamTP). This nucleotide analog causes replicational errors, leading to AT-to-GC transitions during DNA synthesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- N4-aminocytidine is a nucleoside analog with potential applications in cellular biology.
- Understanding the mutagenic mechanisms of such analogs is crucial for their safe and effective use.
Purpose of the Study:
- To investigate the mechanism of mutation induced by N4-aminocytidine in mouse FM3A cells.
- To determine if the metabolite N4-aminodeoxycytidine 5'-triphosphate (dCamTP) is incorporated into DNA during replication and its mutagenic potential.
Main Methods:
- Mouse FM3A cells were treated with N4-aminocytidine to induce mutations.
- In vitro DNA synthesis assays were performed using DNA polymerase alpha and radiolabeled dCamTP.
- Nucleotide incorporation and base pairing fidelity were analyzed using various DNA templates and deoxynucleoside triphosphates.
Main Results:
- N4-aminocytidine treatment induced ouabain resistance mutations in FM3A cells.
- The metabolite dCamTP was incorporated into newly synthesized DNA by DNA polymerase alpha, substituting for dCTP or dTTP.
- Cell-free experiments demonstrated that dCamTP incorporation can lead to AT-to-GC transitions due to mispairing with guanine.
Conclusions:
- The mutagenic effect of N4-aminocytidine in FM3A cells is likely due to replicational errors caused by the incorporation of its metabolite, dCamTP.
- dCamTP incorporation leads to specific base substitutions, highlighting its role in mutagenesis.
- This study provides insights into the molecular mechanisms underlying nucleoside analog-induced mutations.