Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.
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.
Takahashi
M
M
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Nishizawa
M
M
Negishi
K
K
Hanaoka
F
F
Yamada
M A
MA
Hayatsu
H
H
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004257:DNA Polymerase II / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects*; D006860:Hydrogen Bonding; D051379:Mice; D009153:Mutagens; D009154:Mutation / Q000187:drug effects*; D013698:Templates, Genetic
D009153:Mutagens; C038769:N(4)-aminocytidine; D003562:Cytidine; D004247:DNA; D004257:DNA Polymerase II