Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair

Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N (6)-ethenoadenosine (1,N (6)-erA) on translesion synthesis (TLS), mediated by human DNA polymerase eta (hpol eta), and on RNase H2-mediated incision. Mass spectral analysis revealed that 1,N (6)-erA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N (6)-erA. We also show that hpol eta acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N (6)-erA, but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol eta favors the addition of dATP and dGTP opposite 1,N (6)-erA. We also found that RNase H2 recognizes 1,N (6)-erA, but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N (6)-erA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N (6)-erA is incompletely incised by RNase H2.