Fig. 3: DNA discontinuities associated with OF processing defects precede DNA:RNA hybrid accumulation.

a Schematic representation of 5’-flap and R-loop substrates used in in vitro cleavage assays. The length of DNA/RNA fragments (nucleotides, nt), the position of fluorescent labels (blue circles), and the site of Rad27 cleavage are indicated. b Labeled 5′-flaps (left panels) or R-loop substrates (right panels) were incubated with the indicated amounts of either wt, E176A or D129A purified Rad27 proteins (pmol). Cleavage products were visualized following denaturing electrophoresis. The position of uncleaved (90) and cleaved (40/65) fragments are indicated (nucleotides). c, d Cleavage efficiency (% of cleaved products over total labeled fragments; mean ± SD; n = 3) as a function of Rad27 amounts (pmol) for wt, E176A or D129A Rad27 variants. e Amounts of wt Rad27 proteins required to achieve 10% cleavage for 5′-flap and R-loop substrates (obtained from (c, d); mean ± SD; n = 3). f DNA:RNA hybrid detection (DRIP-qPCR; adjusted % of IP; mean ± SD; n = 4; *, p = 0.0286) at the YEF3 locus in RAD27-AID cells (control or auxin-treated) carrying an empty vector (EV) or a construct expressing the Rad27-E176A mutant protein. g Serial dilutions of RAD27-AID cells were grown at the indicated temperatures (30 °C, 37 °C) on rich medium (YPD) in the absence of the presence of auxin. Cells carried an empty vector (EV) or a construct over-expressing Exo1 (pEXO1). h DNA:RNA hybrid detection (DRIP-qPCR; adjusted % of IP; mean ± SD; n = 5; *, p = 0.0317; **, p = 0.0079) at the YEF3 locus in RAD27-AID cells (control or auxin-treated) carrying an empty vector (EV) or a construct over-expressing Exo1. i Serial dilutions of the indicated strains were grown at 30 °C on rich medium (YPD) in the absence or presence of auxin. DNA:RNA hybrid detection (DRIP-qPCR; adjusted % of IP; mean ± SD; n = 4; *, p = 0.0286) at the YEF3 locus in RAD27-AID wt, exo1∆ (j) or cdc9-1 (k) derivatives (control or auxin-treated). The same wt control is used in (j, k). For all DRIP-qPCR assays, when indicated, DNA extracts were treated with RNase H in vitro prior to immunoprecipitation. Statistical test: Two-sided Mann–Whitney–Wilcoxon rank sum test. Source data are provided as a Source Data file.