Figure 4. Highly transcribed units interfere with BIR.

a, P_GAL1-HIS3 or P_TET(on)-HIS3 inserted at MATα-inc or at 6 kb position in two orientations. b, c, AMBER analysis in strains with H-On P_GAL1-HIS3 at MATα-inc (b) and at 6kb (c). d, BIR synthesis negatively correlates with transcription level at P_TET(on)-HIS3. Amount of doxycycline (DOX) added to induce transcription is indicated. Blue, BIR synthesis at 0.5-kb 10hr post-DSB induction; red, mRNA levels of P_TET(on)-HIS3 1hr after addition of doxycycline (just before DSB induction by galactose). b, c, and d, each represents one out of three independent biological repeats that showed similar results (see Supplementary Table 5 for other repeats). Mean values of target to reference (ACT1) loci ratios were calculated by Poisson distribution based on 10,000 droplets with error bars representing upper and lower Poisson 95%CI. e, mRNA level of HIS3 11hr after addition of 5μg/ml doxycycline to cells with or without DSB. The means ± SD (n=3 independent biological repeats) are indicated. Asterisks indicate statistical significance (p=0.0065) determined by t-test (two tailed). f, rPolII enrichment at transcription end site (TES) detected by ChIP-seq for all H-On genes (i), but not in (ii) Co-D genes located on donor ChrIII within 30 kb centromere distal to MAT. X-axis: transcription start site (TSS), TES, and 500 bp flanking regions. Y axis: the mean value of rPolII depth. g, The effect of transcription on BIR-associated mutagenesis measured as Ura⁺ frequency resulting from BIR synthesizing across P_URA3:ura3–29 (“low” transcription) or P_GAL1:ura3–29 (“high” transcription) in either the H-On or Co-D orientation (in respect to BIR) inserted 6 kb centromere distal to the DSB. GAL-HO cut site eliminated in the no-break control strains. The medians and 95% CI of mutation frequencies are indicated (n=6 independent biological repeats). **p=0.0022 determined by the Mann-Whitney U-test (two-tailed). (See Supplementary Table 2).