Abstract
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,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2-mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA 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,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA 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,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.
Highlights
Incorporation of ribonucleotides into DNA can severely diminish genome integrity
We previously reported that hpol can extend an RNA primer [74], and we examined the ability of hpol to extend an RNA primer in the presence of an unmodified as well as a modified ribonucleotide in DNA using physiological concentrations of dNTPs and ribonucleoside triphosphates (rNTPs)
RNTP insertion during DNA replication is a major concern because more than one million rNTPs are incorporated per cell cycle due to their cellular abundance [93]. rNTPs and dNTPs are the main precursors of RNAs and DNAs and can be damaged endogenously in the nucleotide pool
Summary
Incorporation of ribonucleotides into DNA can severely diminish genome integrity. how ribonucleotides instigate DNA damage is poorly understood. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-⑀rA) on translesion synthesis (TLS), mediated by human DNA polymerase (hpol ), and on RNase H2–mediated incision. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol favors the addition of dATP and dGTP opposite 1,N6-⑀rA. Our own work has shown that low-fidelity human DNA polymerase (hpol ) incorporates rATP opposite a sylase; UPLC, ultraperformance liquid chromatography; CID, collisioninduced dissociation; RT, reverse transcriptase; nt, nucleotides.
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