Supplementary Table 1a. Number of crossover (CO), noncrossover (NCO) and breakinduced replication (BIR) events in red/white sectored colonies

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1 Supplementary Table 1a. Number of crossover (CO), noncrossover (NCO) and breakinduced replication (BIR) events in red/white sectored colonies CO NCO BIR Total WT yen rad rad1 yen rad yen rad1 yen Supplementary Table 1b. Fisher s exact test P values between relevant strains for CO events (gray area) or BIR events (white area) in red/white-sectored colonies WT rad1 rad1 yen1 yen1 rad1 WT rad rad1 yen1 yen1 rad

2 Supplementary Table. Yeast strains Strain Genotype* Source or reference LSY0697 MATa met17-s ADE 1 LSY106 MATa met17-s ADE ::KanMX6 LSY1801-4C MATa met17-s ADE :: KanMX6 LSY105-5A MATa met17-s ADE rad1::leu LSY105-6D MATa met17-s ADE rad1::leu ::KanMX6 LSY105-9B MATa met17-s ADE :: KanMX6 rad1::leu LSY108-3C MATa met17-s ADE rad1::leu LSY C MATa met17-s ADE LSY0-10C MATα ade-n his3::natmx4 met::klura3 rad1::leu LSY0-11A MATα ade-n his3::natmx4 met::klura3 ::KanMX6 rad1::leu LSY0-15D MATa ade-n his3::natmx4 met::klura3 LSY0-17B MATα ade-n his3::natmx4 met::klura3 ::KanMX6 rad1::leu LSY0-19D MATα ade-n his3::natmx4 met::klura3 ::KanMX6 LSY0-4C MATa ade-n his3::natmx4 met::klura3 rad1::leu LSY0-6A MATa ade-n his3::natmx4 met::klura3 ::KanMX6 LSY0-4A MATa ade-n his3::natmx4 met::klura3 LSY05-3A ΜΑΤα ade-i lys::gal-iscei his3::hphmx4 rad1::leu LSY05-6A ΜΑΤα ade-i lys::gal-iscei his3::hphmx4 ::KanMX6 LSY05-7C ΜΑΤa ade-i lys::gal-iscei his3::hphmx4 :: KanMX6 rad1::leu LSY05-11C ΜΑΤα ade-i lys::gal-iscei his3::hphmx4 LSY05-11D ΜΑΤa ade-i lys::gal-iscei his3::hphmx4 rad1::leu LSY05-4D ΜΑΤα ade-i lys::gal-iscei his3::hphmx4 LSY05-67B ΜΑΤa ade-i lys::gal-iscei his3::hphmx4 ::KanMX6 rad1::leu LSY05-77B ΜΑΤa ade-i lys::gal-iscei his3::hphmx4 ::KanMX6 LSY199 MATa-inc ura3-hocs lys::ura3-hocs inc 3 (1.kb) ade3::gal-ho LSY540-8A MATa-inc ura3-hocs lys::ura3-hocs inc (1.kb) ade3::gal-ho rad1::leu LSY540-1A MATa-inc ura3-hocs lys::ura3-hocs inc (1.kb) ade3::gal-ho ::KanMX6

3 LSY540-16C LSY540-18A LSY540-19D LSY566-3D LSY566-5A LSY50 LSY51 LSY548-1D LSY548-11A LSY548-9C LSY548-39D LSY548-4A LSY548-66B MATa-inc ura3-hocs lys::ura3-hocs inc (1.kb) ade3::gal-ho MATa-inc ura3-hocs lys::ura3-hocs inc (1.kb) ade3::gal-ho ::KanMX6 MATa-inc ura3-hocs lys::ura3-hocs inc (1.kb) ade3::gal-ho ::KanMX6 rad1::leu MATa-inc ura3-hocs lys::ura3-hocs inc (1.kb) ade3::gal-ho rad1::leu MATa-inc ura3-hocs lys::ura3-hocs inc (1.kb) ade3::gal-ho rad1::leu ::KanMX6 MATa-inc ura3::hocs lys::ura3-hocs inc (5.6 kb) ade3::gal-ho MATa-inc ura3::hocs lys::ura3-hocs inc (5.6 kb) ade3::gal-ho yen1::kanmx6 MATa-inc ura3::hocs lys::ura3-hocs inc (5.6 kb) ade3::gal-ho rad1::leu MATa-inc ura3::hocs lys::ura3-hocs inc (5.6 kb) ade3::gal-ho rad1::leu ::KanMX6 MATa-inc ura3::hocs lys::ura3-hocs inc (5.6 kb) ade3::gal-ho ::KanMX6 MATa-inc ura3::hocs lys::ura3-hocs inc (5.6 kb) ade3::gal-ho ::KanMX6 rad1::leu MATa-inc ura3::hocs lys::ura3-hocs inc (5.6 kb) ade3::gal-ho rad1::leu MATa-inc ura3::hocs lys::ura3-hocs inc (5.6 kb) ade3::gal-ho ::KanMX6 4 4 *All strains are of the W303 genotype (his3-11, 15 leu-3, 11 trp1-1 ade-1 can1-100), only mating type and differences from the standard genotype are listed. References 1 Bartsch, S., Kang, L. E. & Symington, L. S. RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates. Mol Cell Biol 0, (000). Ho, C. K., Mazon, G., Lam, A. F. & Symington, L. S. Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast. Mol Cell 40, (010). 3 Aylon, Y., Liefshitz, B., Bitan-Banin, G. & Kupiec, M. Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae. Mol Cell Biol 3, (003).

4 4 Agmon, N., Yovel, M., Harari, Y., Liefshitz, B. & Kupiec, M. The role of Holliday junction resolvases in the repair of spontaneous and induced DNA damage. Nucleic Acids Res 39, (011).

5 a Untransformed strain: S S 6.7 probe Plasmid integration events: S B S S S B S S B S S B B S b U Δ yen1δ Δ rad1δ yen1δ Supplementary Figure 1. Homologous plasmid integration occurs in the absence of Mus81 and Yen1. a Digestion of genomic DNA with SnaBI (S) and BamHI (B) distinguishes between the untransformed strain (U), random integration and homologous integration at the MET17 locus. Conversion of the chromosomal met17-s allele gives rise to the distinct restriction fragments observed from independent transformants. b Representative transformants with a stable Ura +! phenotype from the Δ yen1δ and Δ rad1δ yen1δ mutants.!!

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8 rad1 yen1 yen1 rad1 rad1 WT White Red Sectored Percent of recombinant colony type Supplementary Figure 4. Percent of each recombinant colony type from the diploid recombination assay. Colonies formed after 1-3 h I-SceI induction were scored as solid red, solid white or red/white sectored in each strain (>500 colonies were scored for each strain from at least three independent trials).

9 Rad1-Rad10 (XPF-ERRC1) > 4nt gap Mus81-Mms4 (MUS81-EME1) < 4nt gap > > Supplementary Figure 5. Preferred substrates for Rad1-Rad10 and Mus81-Mms4 cleavage.!

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