Untargeted Mutation Triggered by Ribonucleoside Embedded in DNA

DNA polymerases frequently misincorporate ribonucleoside 5'-triphosphates into nascent DNA strands. This study examined the effects of an incorporated ribonucleoside on untargeted mutations in human cells. Riboguanosine (rG) was introduced into the downstream region of the <i>supF</i> gene to preferentially detect the untargeted mutations. The plasmid containing rG was transfected into U2OS cells and the replicated DNA was recovered after 48 h. The mutation analysis using the indicator <i>Escherichia coli</i> RF01 strain showed the frequent induction of untargeted base substitutions at the G bases of 5'-GpA-3' dinucleotides, similar to action-at-a-distance mutations induced by an oxidatively damaged base, 8-oxo-7,8-dihydroguanine, and an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) cytosine deaminase. APOBEC3B was then knocked down by RNA interference and the plasmid bearing rG was introduced into the knockdown cells. The untargeted mutations at 5'-<i>G</i>pA-3' sites were reduced by ~80%. These results suggested that ribonucleosides embedded in DNA induce base substitution mutations at G bases in the same strand by an APOBEC3B-dependent mechanism, implying that ribonucleosides contribute to APOBEC3-dependent cancer initiation events.