N
4-Aminocytidine is strongly mutagenic towards E. coli, S. typhimurium, B. subtilis and coliphages φX174 and M13mp2. It also causes mutations in mammalian cell lines and somatic cell mutations in D. melanogaster. The sequence analysis of deoxyribonucleic acid (DNA) from mutated phages revealed that N
4-aminocytidine induces both adeninethymine (AT) to guanine-cytosine (GC) and GC to AT transitions. No transversions are detectable. When E. coli and the mammalian cells were cultured in the presence of [
3H]-N
4-aminocytidine, [
3H]-N
4-aminodeoxycytidine was found in their DNA. It is likely that N
4-aminocytidine is metabolized within the cells into N
4-aminodeoxycytidine 5'-triphosphate (dC
amTP), which is then incorporated into DNA, thereby causing base-pair transitions. To prove this hypothesis, we studied the incorporation of dC
amTP into polynucleotides in the in vitro DNA synthesis catalyzed by E. coli DNA polymerase I large fragment (Klenow enzyme) and DNA polymerase α from a mouse cell line. Both polymerases catalyze incorporation of dC
amTP into DNA efficiently in place of dCTP opposite guanine, and less efficiently, but to a significant extent, in place of dTTP opposite adenine. These observations prove the erroneous nature of dC
amTP as a substrate for DNA synthesis. DNA containing N
4-aminocytosine was prepared by the incorporation of dC
amTP into single-stranded phage DNA annealed to complementary oligonucleotides. The DNA was transfected to E. coli cells. The analysis of progeny phages indicates that N
4-aminocytosine residue in DNA causes A to G or G to A mutation in the position opposite to the site where N
4-aminocytosine should be incorporated.
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