Fig. 1: Ribonucleotide incorporation into DNA in POLE1-M630F and POLA1-Y865F cells.

a Schematic representation of Pu-seq. Top: ribonucleotides (R) are incorporated by the mutated DNA polymerase. Ochre lines indicate the part of DNA synthesis by the mutated polymerase. Middle: in the absence of RNase H2-dependent RER, rNMPs remain in the DNA. Bottom: the sugar backbone of DNA strand is cleaved at sites of rNMP incorporation by alkali. Small ssDNA fragments (dashed box) are collected and subjected to library preparation and sequencing to identify the 5ʹ end of ssDNA fragment (circled end of ochre lines) as the location of rNMP. b Left: auxin-induced degradation of RNASEH2A following addition of indole-3-acetic acid (IAA) to cells expressing wild type O. sativa TIR1 (AID system). Right: 5-Ph-IAA-induced degradation of RNASH2A in cells expressing O. sativa TIR1(F74G) (AID2 system). We repeated this experiment three times and obtained similar results. c Conservation of targeted amino acid residues of DNA Polε and Polα to induce rNMP incorporation. d–f Determination of rNMP incorporation into genomic DNA. Extracted genomic DNA from the indicated cell lines after the initiation of RNASEH2A degradation was treated with alkali to cleave at incorporated rNMP and analysed by electrophoresis. Source data of (b–f) are provided as a Source Data file. We repeated experiments in (b–f) three times and obtained similar results (see replicated results of (d) and (f) in Supplementary Fig. 1a).