Tracking break-induced replication shows that it stalls at roadblocks

Abstract

Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human diseases1,2. Previous studies have defined the enzymes that are required for BIR1,2,3,4,5; however, understanding of initial and extended BIR synthesis, and of how the migrating D-loop proceeds through known replication roadblocks, has been precluded by technical limitations. Here we use a newly developed assay to show that BIR synthesis initiates soon after strand invasion and proceeds more slowly than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but is unable to proceed beyond 30 kilobases, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32, but does not proceed efficiently. Interstitial telomeric DNA disrupts and terminates BIR progression, and BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and how BIR contributes to genome instability.

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Fig. 1: The dynamics, efficiency and rate of BIR synthesis determined by AMBER.
Fig. 2: Primase is required to stabilize long leading strand ssDNA.
Fig. 3: Interstitial telomere sequence interrupts BIR.
Fig. 4: Highly transcribed units interfere with BIR.

Data availability

The whole-genome sequencing raw data have been deposited to the NCBI Sequence Read Archive database under bioproject accession PRJNA671607. The rPolII ChIP sequencing data have been deposited to the NCBI Gene Expression Omnibus under accession GSE159384. All data generated and analysed in this paper are available from the corresponding author upon request.

References

  1. 1.

    Anand, R. P., Lovett, S. T. & Haber, J. E. Break-induced DNA replication. Cold Spring Harb. Perspect. Biol. 5, a010397 (2013).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Llorente, B., Smith, C. E. & Symington, L. S. Break-induced replication: what is it and what is it for? Cell Cycle 7, 859–864 (2008).

    CAS  PubMed  Google Scholar 

  3. 3.

    Sakofsky, C. J. & Malkova, A. Break induced replication in eukaryotes: mechanisms, functions, and consequences. Crit. Rev. Biochem. Mol. Biol. 52, 395–413 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Lydeard, J. R., Jain, S., Yamaguchi, M. & Haber, J. E. Break-induced replication and telomerase-independent telomere maintenance require Pol32. Nature 448, 820–823 (2007).

    ADS  CAS  PubMed  Google Scholar 

  5. 5.

    Wilson, M. A. et al. Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration. Nature 502, 393–396 (2013).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Malkova, A., Naylor, M. L., Yamaguchi, M., Ira, G. & Haber, J. E. RAD51-dependent break-induced replication differs in kinetics and checkpoint responses from RAD51-mediated gene conversion. Mol. Cell. Biol. 25, 933–944 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Saini, N. et al. Migrating bubble during break-induced replication drives conservative DNA synthesis. Nature 502, 389–392 (2013).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Donnianni, R. A. & Symington, L. S. Break-induced replication occurs by conservative DNA synthesis. Proc. Natl Acad. Sci. USA 110, 13475–13480 (2013).

    ADS  CAS  PubMed  Google Scholar 

  9. 9.

    Lydeard, J. R. et al. Break-induced replication requires all essential DNA replication factors except those specific for pre-RC assembly. Genes Dev. 24, 1133–1144 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Ruff, P., Donnianni, R. A., Glancy, E., Oh, J. & Symington, L. S. RPA stabilization of single-stranded DNA is critical for break-induced replication. Cell Rep. 17, 3359–3368 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Piazza, A., Koszul, R. & Heyer, W. D. A proximity ligation-based method for quantitative measurement of D-loop extension in S. cerevisiae. Methods Enzymol. 601, 27–44 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Piazza, A. et al. Dynamic processing of displacement loops during recombinational DNA repair. Mol. Cell 73, 1255–1266.e4 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Jain, S. et al. A recombination execution checkpoint regulates the choice of homologous recombination pathway during DNA double-strand break repair. Genes Dev. 23, 291–303 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Raghuraman, M. K. et al. Replication dynamics of the yeast genome. Science 294, 115–121 (2001).

    ADS  CAS  PubMed  Google Scholar 

  15. 15.

    Smith, C. E., Llorente, B. & Symington, L. S. Template switching during break-induced replication. Nature 447, 102–105 (2007).

    ADS  CAS  PubMed  Google Scholar 

  16. 16.

    Anand, R., Beach, A., Li, K. & Haber, J. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates. Nature 544, 377–380 (2017).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Donnianni, R. A. et al. DNA polymerase delta synthesizes both strands during break-induced replication. Mol. Cell 76, 371–381.e4 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Francesconi, S. et al. Mutations in conserved yeast DNA primase domains impair DNA replication in vivo. Proc. Natl Acad. Sci. USA 88, 3877–3881 (1991).

    ADS  CAS  PubMed  Google Scholar 

  19. 19.

    Aksenova, A. Y. & Mirkin, S. M. At the beginning of the end and in the middle of the beginning: structure and maintenance of telomeric DNA repeats and interstitial telomeric sequences. Genes 10, E118 (2019).

    PubMed  Google Scholar 

  20. 20.

    Kim, N. & Jinks-Robertson, S. Guanine repeat-containing sequences confer transcription-dependent instability in an orientation-specific manner in yeast. DNA Repair 10, 953–960 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Piazza, A. et al. Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae. Nucleic Acids Res. 38, 4337–4348 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Geronimo, C. L. & Zakian, V. A. Getting it done at the ends: Pif1 family DNA helicases and telomeres. DNA Repair 44, 151–158 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Beck, C. R. et al. Megabase length hypermutation accompanies human structural variation at 17p11.2. Cell 176, 1310–1324.e10 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Hu, Q. et al. Break-induced replication plays a prominent role in long-range repeat-mediated deletion. EMBO J. 38, e101751 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Li, S. & Wu, X. Common fragile sites: protection and repair. Cell Biosci. 10, 29 (2020).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Macheret, M. et al. High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing. Cell Res. 30, 997–1008 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Costantino, L. & Koshland, D. Genome-wide map of R-loop-induced damage reveals how a subset of R-loops contributes to genomic instability. Mol. Cell 71, 487–497.e3 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Minocherhomji, S. et al. Replication stress activates DNA repair synthesis in mitosis. Nature 528, 286–290 (2015).

    ADS  CAS  PubMed  Google Scholar 

  29. 29.

    Johnston, M. & Davis, R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 1440–1448 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Batrakou, D. G., Heron, E. D. & Nieduszynski, C. A. Rapid high-resolution measurement of DNA replication timing by droplet digital PCR. Nucleic Acids Res. 46, e112 (2018).

    PubMed  PubMed Central  Google Scholar 

  31. 31.

    Deem, A. et al. Defective break-induced replication leads to half-crossovers in Saccharomyces cerevisiae. Genetics 179, 1845–1860 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Holmes, A. M. & Haber, J. E. Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases. Cell 96, 415–424 (1999).

    CAS  PubMed  Google Scholar 

  33. 33.

    Morawska, M. & Ulrich, H. D. An expanded tool kit for the auxin-inducible degron system in budding yeast. Yeast 30, 341–351 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Storici, F. & Resnick, M. A. Delitto perfetto targeted mutagenesis in yeast with oligonucleotides. Genet. Eng. (N. Y.) 25, 189–207 (2003).

    CAS  Google Scholar 

  35. 35.

    Griffith, J., Bianchi, A. & de Lange, T. TRF1 promotes parallel pairing of telomeric tracts in vitro. J. Mol. Biol. 278, 79–88 (1998).

    CAS  PubMed  Google Scholar 

  36. 36.

    Tian, M. & Alt, F. W. Transcription-induced cleavage of immunoglobulin switch regions by nucleotide excision repair nucleases in vitro. J. Biol. Chem. 275, 24163–24172 (2000).

    CAS  PubMed  Google Scholar 

  37. 37.

    Yadav, P. et al. Topoisomerase I plays a critical role in suppressing genome instability at a highly transcribed G-quadruplex-forming sequence. PLoS Genet. 10, e1004839 (2014).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Vaze, M. B. et al. Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase. Mol. Cell 10, 373–385 (2002).

    CAS  PubMed  Google Scholar 

  39. 39.

    Elango, R., Kockler, Z., Liu, L. & Malkova, A. in Investigation of Break-Induced Replication in Yeast. Methods in Enzymology Vol. 601, 161–203 (Elsevier, 2018).

  40. 40.

    Deem, A. et al. Break-induced replication is highly inaccurate. PLoS Biol. 9, e1000594 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Sakofsky, C. J. et al. Translesion polymerases drive microhomology-mediated break-induced replication leading to complex chromosomal rearrangements. Mol. Cell 60, 860–872 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Elango, R. et al. Repair of base damage within break-induced replication intermediates promotes kataegis associated with chromosome rearrangements. Nucleic Acids Res. 47, 9666–9684 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Chung, W. H., Zhu, Z., Papusha, A., Malkova, A. & Ira, G. Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting. PLoS Genet. 6, e1000948 (2010).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Genomic and RNA Profiling Core at Baylor College of Medicine for library preparation and NGS service; Y. Pavlov for advice on western blot analysis; J. Haber for sharing plasmids and yeast strains ; N. Kim for sharing the plasmids containing G4-forming sequences; P. Sung for the gift of the anti-Rad51 antibody; C. Nieduszynski and Marc Wold for suggestions; and S. Smolikove for comments on the manuscript. This work was supported by NIH grants R35GM127006 to A.M., GM080600, GM125650 to G.I., and R01AG052507 to W.D.

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Contributions

L.L., Z.Y., G.I., and A.M. designed the study. L.L. performed all AMBER analysis, and the majority of strain construction and genetic analysis. Z.Y. performed all ChIP assays. B.A.O. conducted experiments aimed to determine the effect of pol3-01 and G4 sequences in BIR. J.T. conducted all CHEF electrophoresis gel analysis and probing and assisted in genetic experiments. L.S. and W.D. analysed the results of ChIP–seq experiments. J.K. conducted S1 treatment in the pri2-1 experiment, data analysis for whole-genome sequencing and mRNA extraction. R.L. constructed several strains including the rrm3Δ and pif1Δ mutants and assisted in genetic experiments. S.K. constructed the strain containing (ITS)~40 and performed its initial characterization. R.E. constructed the pol1-AID and pol2-AID strains. H.L. performed western blot analyses. L.L., Z.Y., J.K., L.S., W.D., B.A.O., R.L., J.T., G.I., and A.M. analysed the results of experiments. L.L., G.I., and A.M. wrote the manuscript.

Corresponding authors

Correspondence to Grzegorz Ira or Anna Malkova.

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Extended data figures and tables

Extended Data Fig. 1 Kinetics of DSB repair.

a, Strand invasion measured by Rad51 ChIP followed by qPCR. Forward primer is shared by donor and recipient at X region, and reverse primer is unique to the region in the donor. The means ± s.d. (n = 3 independent biological repeats) are indicated. b, Strand invasion measured by DLC assay. DLC signal is normalized to ARG4 and then compared to 0hr by fold changes. The means ± s.d. (n = 3 independent biological repeats) are indicated. c, DNA synthesis detected by ddPCR using traditional DNA preparation protocol as previously described39. d, AMBER analysis of DNA clipping or degradation using primers located centromere proximal to MATa in wt (top), rad51Δ (middle), or rad52Δ (bottom) following DSB induction. In rad52Δ the rate of DNA degradation was >8kb/hr, much higher than in wt and rad51Δ. c and d each represents one out of three independent biological repeats that showed similar results (see Supplementary Table 6 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.

Extended Data Fig. 2 Kinetics of BIR synthesis.

a, Schematic of primers (triangles) used for AMBER analyses (similar to Fig. 1a). b, DNA synthesis detected by AMBER in a time course designed as described in Fig. 1b, but with nocodazole addition 3 h before DSB induction (0hr). The data represent one experiment that was similar to 3 experiments including the one presented in Fig. 2b. c, Slopes of BIR at the time when 50 percent of the cells had completed BIR (V50) at the indicated chromosomal position based on three independent experiments (n = 3), including the one shown in Fig. 1b. The means ± s.d. (error-bars) are indicated. Statistics is determined by two-tailed t-test with p value indicated. d, AMBER analysis of POL3 (wt), and e, pol3-01. b, d and e each represents one out of three independent biological repeats that showed similar results (see Supplementary Table 6 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.

Extended Data Fig. 3 BIR synthesis in pif1Δ, pol32Δ, and pol2-AID.

a, Strand invasion in pif1Δ and pol32Δ mutants 6h post DSB measured by DLC assay. DLC signal is normalized to ARG4 and compared to 0hr by fold changes. The means ± s.d. (n = 3 independent biological repeats) are indicated. b, Kinetics of Ya disappearance in wt, pif1Δ, pol32Δ and pif1Δ pol32Δ. c, Schematic of primers used for AMBER analyses. df, AMBER analysis of BIR synthesis in wt (PIF1, POL32) (d), in pif1Δ (e), and pol32Δ (f). d-f each represents one out of three independent biological repeats that showed similar results (see Supplementary Table 6 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. g, AMBER analysis of BIR synthesis following inactivation of Polε (pol2-degron) with schematic of analysis (top), degradation of AID-tagged Pol2 verified by Western Blot (middle) and calculation of copy number (bottom). The means ± s.d. (n = 3 independent biological repeats) are indicated. * represents significant difference (P = 0.0351) determined by t-test, two-tailed).

Extended Data Fig. 4 Primase defect leads to defective long leading strand synthesis.

a, CHEF gel electrophoresis analysis of DSB repair in PRI2 (wt) and temperature-sensitive pri2-1 mutant followed by Southern blot analysis using ADE1-specific probe (one representative experiment from n = 3 independent biological repeats). BIR: BIR repair product; HC: half-crossover resulting from fusion between fragments of recipient and donor Chromosome III (see Extended Data Fig. 9a for details); ChrI: Chromosome I; Uncut: full-length recipient chromosome. b, Top: Schematic of experiment to characterize BIR progression following depletion of Pol1 (pol1-degron) by addition of IAA. Bottom left: Degradation of AID-tagged Pol1 verified by Western Blot. Bottom right: AMBER analysis of BIR synthesis. The means ± s.d. (n = 3 independent biological repeats) indicated by error bars. c, Proposed model explaining the role of primase and Okazaki fragment synthesis in stabilization of leading strand.

Extended Data Fig. 5 BIR stalls inside ITS.

a, AMBER analysis of BIR synthesis in TLC1 and tlc1Δ cells traversing (ITS)~40, similar to Fig. 3b, c, but with nocodazole added 6hr post DSB. The means ± s.d. (n = 3 independent biological repeats) are indicated. **Statistical difference is determined by t-test (two-tailed), P = 0.0053. b, Proposed model of BIR interruption at ITS followed by telomerase-mediated de novo telomere addition.

Extended Data Fig. 6 G4 -forming sequence does not block BIR progression.

a, A G4-forming sequence or (TTAGGG)28 was inserted at the 6-kb position. The colours indicating positions are kept the same in panels b, d, e, g and h. b, AMBER analysis of BIR through (ITS)28 inserted at the 6 kb position. c, Sanger sequencing analysis of repair outcomes of BIR that traversed (ITS)28 show change in ITS copy number resulting from template switching. (Sample sizes (n) are indicated. * = statistically significant difference (P = 0.0288) determined by Chi-square test (two-sided, df = 1)). d, AMBER analysis of BIR through sequence that can form G4 structure during leading strand (Ori1) synthesis and e, during lagging strand (Ori2) synthesis. f, AMBER analysis of BIR through G4-forming sequences similar to d and e, but in the presence of G4-stabilizing drug, Phen-DC3. The means ± s.d. (n = 3 independent biological repeats) indicated by error-bars. g, h, AMBER analysis of BIR synthesis in rrm3Δ mutant through (ITS)28 (g) and through (ITS)~40 (h). i, genetic analysis of GCRs in (g) and (h) performed similar to Fig. 3d, e. Sample sizes (n) are indicated. b, d, e, g and h each represents one out of three independent biological repeats that showed similar results (see Supplementary Table 6 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.

Extended Data Fig. 7 Initiation of BIR immobilizes rPolII on H-On transcribed genes.

a, Location of primer pairs homologous to GAL1 promoter (pair 1), TSS (transcription start site) (pair 2) and the middle of HIS3 gene (pair 3) used for qPCR (see Supplementary Table 4 for primer sequences). b, RNA PolII enrichment at PGAL1-HIS3 in H-On and Co-D orientations measured at indicated times following galactose addition. The means of rPolII enrichment ± s.d. indicated by error-bars (n = 3 independent biological experiments). c, Transcription levels of PGAL1-HIS3 8 h post-DSB detected by HIS3-specific primers and normalized to ACT1 mRNA control. The means ± s.d. (n = 3 independent biological repeats), indicated by error-bars. (** = statistically significant difference (P = 0.0033) determined by t-test, two tailed).

Extended Data Fig. 8 Interference between BIR and transcription.

a, Schematic of PGAL1-HIS3 inserted at MATα-inc in H-On or Co-D orientations with respect to BIR progression. The same primer pairs were used for AMBER analysis as described in Fig. 1a, although their actual positions along the BIR track (donor chromosome) are shifted by insertion of PGAL1-HIS3. b, AMBER analysis of experiment in Fig. 4b using primers located in HIS3 gene. c, Strand invasion kinetics for experiment shown in d and Fig. 4b assessed by Rad51 ChIP followed by qPCR using primers amplifying the junction region with the forward primer targeting a donor and recipient shared region while the reverse primer targeting donor-specific region. The means ± s.d. (n = 3 independent biological repeats) are indicated. d, AMBER analysis of AM1411 (the NO-PGAL1-HIS3 control strain used for experiments shown in Fig. 4b, and Extended Data Fig. 8e) containing insertion of lys2 under its native promoter at MATα-inc to match the experimental strains in mating type and in the presence of insertion at MATα-inc. e, AMBER analysis in strain with PGAL1-HIS3 inserted at MATα-inc in Co-D orientation. f, The amount of BIR synthesis detected at 10 h using 0.5 kb primers in strains with or without PGAL1-HIS3. The means ± s.d. (n = 3 independent biological repeats) are indicated. Asterisks indicate statistically significant difference determined by two-tailed t-test (***: P = 1.12e-4; **: P = 0.0035) g, AMBER analysis of BIR progression in strains with PGAL1-HIS3 inserted 6 kb centromere distal from MAT in Co-D orientation. b, d, e, and g each represents one out of three independent biological repeats that showed similar results (see Supplementary Table 6 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.

Extended Data Fig. 9 BIR is interrupted by transcription.

a, Products of DSB repair as distinguished by genetic markers. b, Distribution of DSB repair outcomes for strains with and without PGAL1-HIS3 inserted at MATα-inc. c, the same as in b, but for strains with PGAL1-HIS3 inserted at 6kb position. In b and c, sample sizes (from 3 biological repeats) are indicated (see Methods for details). Statistical comparisons are performed using Chi-square test (two-sided, df = 1); p values are indicated. d, (i) rPolII enrichment at TES detected by ChIP-seq for PWP2 gene located 20kb centromere distal to MAT. Red rectangle: BIR-promoted accumulation of rPolII at TES; (ii) rPolII distribution for all H-On genes located on donor chromosome III between 60 and 90 kb centromere distal to MAT. Data from the same experiment as shown in Fig. 4f. The labels are similar to Fig. 4f. e, AMBER analysis of BIR synthesis from the same samples as in Fig. 4f (n = 1, mean values of ratios between target and reference (ACT1) loci are calculated using Poisson distribution based on 10,000 droplets with error bars representing upper and lower Poisson 95%CI.). f, independent repeat of experiment shown in Fig. 4f. Metagene plot showing the distribution of normalized rPolII-ChIP-seq reads along chromosome III. i) At genes located within 0-30kb centromere distal of the MAT locus (n = 10) in H-On direction; ii) at genes located within 60-90kb centromere distal to the MAT locus in H-On orientation (n = 11); iii) at genes located within 0-30kb centromere distal to the MAT locus in Co-D orientation (n = 9); iv) at all the genes except genes on chromosome III (n = 2950). Transcription start site (TSS), transcription end site (TES) and 500 bp flanking regions were plotted. Y axis showing the mean value of rPolII depth. g, Example of BIR-caused rPolII re-distribution in yeast gene PWP2 located on chrIII (data from same experiment in Extended Data Fig. 9f). The panels show normalized rPolII ChIP-seq reads from RAD51 (wild type) (top) and rad51Δ (bottom). Red rectangle: BIR-promoted rPolII accumulation.

Extended Data Fig. 10 Model of highly transcribed region interfering BIR.

a, Model of the block of BIR initiation by transcription. Left: For H-On transcription, BIR immobilizes rPolII, which leads to the block of BIR and transcription. Right: Co-D transcription also interferes with BIR initiation. b, Established BIR can traverse transcription units in both orientations, even though it is associated with transient accumulation of rPolII at TES and leads to increased level of GCRs and mutagenesis in H-On orientation.

Supplementary information

Supplementary Figure

This file contains Supplementary Figure 1: Original Source Images. a-e, Source images for Fig. 3d (a), Extended Data. Fig. 3g (b), 4a (c, e) and 4b (d). Boxed parts are shown in the Figures or Extended Data figures. f-i, Analysis of outcomes for Extended Data Fig. 6i (f), 9c (g), 9b (h), and Supplemental Table 1 (g, h)

Reporting Summary

Supplementary Table

Supplementary Table 1: The level of GCR following the passage of high-transcription units during BIR vs normal replication.

Supplementary Table

Supplementary Table 2: The effect of transcription on mutagenesis associated with BIR.

Supplementary Table

Supplementary Table 3. Yeast strain list.

Supplementary Table

Supplementary Table 4. List of primers used for qPCR, and ddPCR experiments.

Supplementary Table

Supplementary Table 5: Data for all three independent biological repeats for experiments presented in Figures (Fig. 1, 2, 3 and 4).

Supplementary Table

Supplementary Table 6: AMBER analysis of all three independent biological repeats for experiments presented in Extended Data.

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Liu, L., Yan, Z., Osia, B.A. et al. Tracking break-induced replication shows that it stalls at roadblocks. Nature 590, 655–659 (2021). https://doi.org/10.1038/s41586-020-03172-w

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