Figure 5. Genetic analysis of the repair and DNA-RNA hybrid accumulation at endonuclease-induced DSBs.

(a) Scheme of the pCM189-L2FHO recombination system. (b) Frequency of HO-induced recombination in wild type (WS), rnh1Δ rnh201Δ (WSR1R2) and hpr1Δ (WSHPR1) strains transformed with pTINV-FHO under low or high transcription (n≥3). (c) Frequency of HO-induced recombination in wild type (WS) and hpr1Δ (WSHPR1) strains transformed with either pRS314 (RH−) or pRS314-GALRNH1 (RH+) and pTINV-FHO under low or high transcription (n=9). (d) DRIP with the S9.6 antibody in the leu2-HO allele as depicted on top and in either spontaneous conditions (HO−) or after HO induction (HO+) in wild type (WS) and rnh1Δ rnh201Δ (WSR1R2) strains transformed with pTINV-FHO under high transcription and either non-treated (RH−) or after in vitro RNase H treatment (RH+) (n=5). (e) DRIP with the S9.6 antibody in the leu2-HO allele as depicted on top and in either spontaneous conditions (HO−) or after HO induction (HO+) in wild type (WS) and rnh1Δ rnh201Δ (WSR1R2) strains transformed with pTINV-FHO under low transcription and either non-treated (RH−) or after in vitro RNase H treatment (RH+) (n=4). (f) DRIP with the S9.6 antibody in the PDC1 gene as depicted on top and in either spontaneous conditions (HO−) or after HO induction (HO+) in wild type (WS) and rnh1Δ rnh201Δ (WSR1R2) strains transformed with pTINV-FHO under high transcription and either untreated (RH−) or after in vitro RNase H treatment (RH+) (n=5). Mean and SEM of independent experiments consisting in the median value of six independent colonies each are plotted in (b–f) panels. *p≤0.05; **p≤0.01; ***p≤0.001 (unpaired Student’s t-test in (b) panel and paired Student’s t-test in (c–f) panels). See also Figure 5—figure supplement 1. Data underlying this figure are provided as Figure 5—source data 1. DRIP, DNA-RNA immunoprecipitation; HR, homologous recombination; trx, transcription.

Figure 5—figure supplement 1. Frequency of recombination and hybrid accumulation upstream of the HO site.

Related to Figure 5. (a) Frequency of spontaneous recombination in wild type (WS), rnh1Δ rnh201Δ (WSR1R2) and hpr1Δ (WSHPR1) strains transformed with pTINV-FHO under low or high transcription (n≥2). (b) DRIP with the S9.6 antibody in the leu2-HO allele as depicted on top and in either spontaneous conditions (HO−) or after HO induction (HO+) in wild type (WS) and rnh1Δ rnh201Δ (WSR1R2) strains transformed with pTINV-FHO under high transcription and either non-treated (RH−) or after in vitro RNase H treatment (RH+) (n=5). Mean and SEM are plotted in all panels. *p≤0.05; **p≤0.01 (paired Student’s t-test). Data underlying this figure are provided as Figure 5—figure supplement 1—source data 1. DRIP, DNA-RNA immunoprecipitation; trx, transcription.