Srs2 helicase is known to dismantle nucleofilaments of Rad51 recombinase to prevent spurious recombination events1,2,3,4,5,6and unwind trinucleotide sequences that are prone to hairpin formation7. Here we document a new, unexpected genome maintenance role of Srs2 in the suppression of mutations arising from mis-insertion of ribonucleoside monophosphates during DNA replication. In cells lacking RNase H2, Srs2 unwinds DNA from the 5′ side of a nick generated by DNA topoisomerase I8at a ribonucleoside monophosphate residue. In addition, Srs2 interacts with and enhances the activity of the nuclease Exo1, to generate a DNA gap in preparation for repair. Srs2–Exo1 thus functions in a new pathway of nick processing-gap filling that mediates tolerance of ribonucleoside monophosphates in the genome. Our results have implications for understanding the basis of Aicardi–Goutières syndrome, which stems from inactivation of the human RNase H2 complex9.