Origins and removal of ribonucleotides in DNA. (A) Incorporation and removal of ribonucleotides incorporated by RNA primase during OF synthesis and maturation. Two of the pathways by which the RNA primer synthesized by Pol α-primase is removed are displayed. RNase H2 is able to digest and remove the RNA primer up until the terminal 3′ ribonucleotide that is removed by FEN1. Alternatively, Pol δ can perform strand displacement synthesis to generate a flap that is degraded by FEN1. (B) Proofreading efficiency is determined by the balance between excision and extension. The polymerase exonuclease activity can excise ribonucleotides from the 3′ terminus in primer-template DNA, an event that is more efficient on a mismatched 3′ terminal dNMP primer than it is on an rNMP. The kinetics of proofreading (both excision and extension) by a replicative DNA polymerase in the presence of a terminal rNMP remain to be biochemically determined. (C) The ribonucleotide excision repair (RER) pathway is initiated by incision 5′ to the ribonucleotide by RNase H2. Strand displacement synthesis by the polymerase is followed by nucleolytic flap processing and ligation. There are redundant functions of the polymerases (Pol δ and Polε) as well as the nucleases (FEN1 and Exo1; Adapted from Fig. 4 in []). (D) Loss of RNase H2 activity due to deletion of RNH201 or RNase H2-deficiency in AGS leaves unrepaired genomic ribonucleotides that may become targets for Top1-cleavage and removal. The resulting unligatable DNA ends possess a 2′–3′-cyclic phosphate and a 5′-hydroxyl that may promote the formation of DNA breaks and/or recombination and cause genome instability. (Adapted from Fig. 4 in []). (E) The pathway by which RNase H2 cleavage at ribonucleotides can generate abortive ligation intermediates (adenylated 5′-RNA–DNA junctions) that require APTXHNT3 for resolution. Failure of this pathway due to deletion of HNT3 or APTX deficiency in AOA1 causes phenotypes that may reflect the persistence of adenylated RNA–DNA junctions (Adapted from Fig. 2E in [].
