Supplementary Figure 1. Representative controls for DSB induction efficiency in wild-type

Supplementary Figure 1. Representative controls for DSB induction efficiency in wild-type and mutant cells. Representative spotting assay reveals that the tested deletions do t affect the efficiency of DSB induction, which results in the loss of the URA3 marker gene. Shown are ten-fold serial dilutions of cells with indicated getypes onto n-selective media (SC), negative selection media (SC-URA; DSB induction indicates less growth), and positive selection media (SC+5FOA; DSB induction indicates more growth). DSB efficiency is consistently above 99.9% in all strains tested. S1

Supplementary Figure 2. ChIP assessing chromatin silencing near the subtelomeric DSB site. Representative anti-diach3 (K9, K14) ChIP gels revealing that the deletion of Lrs4, but t Cik1 or Nup84, greatly compromises silent chromatin assembly near the uninduced DSB site. Relative fold enrichments for regions surrounding the DSB site are presented below the gel. Results are rmalized to ACT1 and presented relative to wild-type cells. S2

Supplementary Figure 3. DSB induction efficiency is similar between cells with or without zip/control sequences integrated near the subtelomeric DSB site. Representative spotting assays indicating that the tested zip/control deletions do t affect the efficiency of subtelomeric DSB induction in wild-type, lrs4, nup84, and cik1 cells. Shown are ten-fold serial dilutions of cells with indicated getypes onto n-selective media (SC) or negative selection media (SC- URA; DSB induction indicates less growth). DSB efficiency is consistently above 99.9% in all strains tested. S3

Supplementary Figure 4. ChIP assessing chromatin silencing near the subtelomeric DSB sites in the presence of zip or control sequences. Representative anti-diach3 (K9, K14) ChIP gels revealing that the deletion of Lrs4, but t Cik1 or Nup84, greatly compromises silent chromatin assembly near the uninduced DSB site. Relative fold enrichments for a region near the DSB site are presented below the gel. Results are rmalized to ACT1 and presented relative to wild-type control. S4

Supplementary Figure 5. Raw data for Western blots shown as cropped images in the main figures. Since the Western blots shown in the main figures are heavily cropped for presentation purposes as a space-saving measure, original larger images of developed films are shown here. The related main figure panel numbers are indicated above each film and the approximate cropped areas shown in the main figure panels are highlighted with dashed boxes. S5

Supplementary Table 1. Relative survival rates of TELXI-L subtelomeric DSB. Getype a,b Rate of survival c P value d Rate relative to WT WT 0.073 (± 0.016) N/A 1.00 nup84 0.035 (± 0.004) <0.0001 0.48 nup145c 0.043 (± 0.005) <0.0001 0.59 esc1 0.036 (± 0.010) <0.0001 0.49 lrs4 0.035 (± 0.011) <0.0001 0.48 csm1 0.032 (± 0.013) <0.0001 0.44 lrs4 csm1 0.035 (± 0.017) <0.0001 0.48 lrs4 esc1 0.039 (± 0.006) <0.0001 0.53 nup84 esc1 0.036 (± 0.007) <0.0001 0.49 nup84 lrs4 0.034 (± 0.008) <0.0001 0.47 heh1 0.054 (± 0.024) 0.1 0.74 nur1 0.069 (± 0.017) 0.64 0.95 cik1 0.031 (± 0.013) <0.0001 0.42 lrs4 cik1 0.035 (± 0.014) <0.0001 0.48 kar3 0.021 (± 0.007) <0.0001 0.29 lrs4 kar3 0.02 (± 0.003) <0.0001 0.27 cin8 0.048 (± 0.005) <0.0001 0.66 bub2 0.068 (± 0.013) 0.51 0.93 rad52 0.008 (± 0.008) <0.0001 0.11 lrs4 rad52 0.006 (± 0.002) <0.0001 0.08 dnl4 0.077 (± 0.008) 0.51 1.05 lrs4 dnl4 0.037 (± 0.008) <0.0001 0.51 ku70 0.08 (± 0.035) 0.65 1.10 pol32 0.004 (± 0.001) <0.0001 0.05 dpb3 0.075 (± 0.010) 0.77 1.03 sae2 0.12 (± 0.033) <0.05 1.64 sir4 0.39 (± 0.107) <0.0001 5.34 sir3 0.45 (± 0.096) <0.0001 6.16 sir3 rad52 0.007 (± 0.003) <0.0001 0.10 slx8 0.015 (± 0.003) <0.0001 0.21 slx5 0.017 (± 0.004) <0.0001 0.23 rad9 0.01 (± 0.003) <0.0001 0.14 swr1 0.048 (± 0.003) <0.0001 0.66 cik1 swr1 0.026 (± 0.005) <0.0001 0.36 tub3 0.038 (± 0.017) <0.0001 0.52 cik1 tub3 0.019 (± 0.003) <0.0001 0.26 a WT and/or cik1 rates are shown in Figs. 1b, 1f, 1g, and 4d for comparison. b Rates in Fig. 1c are repeated from Fig. 1b with superimposed colour-coded results of PCR survival analysis. c Standard deviations are shown between brackets. d P calculated using Student's t-test. S6

Supplementary Table 2. Relative survival rates of internal DSB on chromosome XI. Getype a Rate of survival b P value c Rate relative to WT WT 0.0023 (± 0.00093) N/A 1.00 rad52 0.0023 (± 0.00069) 0.99 1.00 ku70 0.00003 (± 0.000005) <0.0001 0.01 dnl4 0.00003 (± 0.00002) <0.0001 0.01 nup84 0.00202 (± 0.00067) 0.59 0.88 esc1 0.00184 (± 0.00064) 0.34 0.80 lrs4 0.00218 (± 0.00065) 0.83 0.95 csm1 0.00271 (± 0.00087) 0.39 1.18 heh1 0.00206 (± 0.00020) 0.53 0.90 nur1 0.00214 (± 0.00029) 0.73 0.93 cik1 0.0019 (± 0.00042) 0.33 0.83 sir4 0.00004 (± 0.00001) <0.0001 0.02 sir3 0.00003 (± 0.000005) <0.0001 0.01 slx8 0.00122 (± 0.00031) <0.05 0.53 slx5 0.00101 (± 0.00017) <0.05 0.44 a WT rates are shown in Figs. 1f and 1g for comparison. b Standard deviations are shown between brackets. c P calculated using Student's t-test. S7

Supplementary Table 3. List of strains used in this study. KMY# Original getype feature pkm97 KMY574 1 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa KMY602 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa + pkm97 [I-Sce I (gal induc), LEU2d, TRP1] KMY778 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nup84δ::kanr KMY827 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nup84δ::kanr KMY575 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nup145cδ::kanr KMY669 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nup145cδ::kanr KMY730 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, esc1δ::hphr KMY756 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, esc1δ::hphr KMY631 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr KMY658 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr KMY663 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, csm1δ::kanr KMY678 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, csm1δ::kanr KMY1583 KMY1663 KMY1142 KMY1152 KMY1145 KMY1196 KMY1121 KMY1139 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, csm1δ::kanr, lrs4 ::HphR MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, csm1δ::kanr, lrs4 ::HphR MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr, esc1δ::hphr MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr, esc1δ::hphr MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nup84δ::kanr, esc1δ::hphr MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nup84δ::kanr, esc1δ::hphr MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nup84δ::kanr, lrs4δ::hphr MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nup84δ::kanr, lrs4δ::hphr KMY666 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, heh1δ::kanr KMY682 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, heh1 ::KanR KMY624 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nur1δ::kanr KMY653 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, nur1δ::kanr KMY838 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cik1δ::kanr KMY882 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cik1δ::kanr KMY1747 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cik1δ::kanr, lrs4 ::HphR S8

KMY1770 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cik1δ::kanr, lrs4 ::HphR KMY1744 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, kar3δ::hphr KMY1764 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, kar3δ::hphr KMY1745 KMY1767 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr, kar3 ::HphR MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr, kar3 ::HphR KMY1617 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cin8δ::hphr KMY1629 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cin8δ::hphr KMY2247 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, bub2δ::natr KMY2272 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, bub2δ::natr KMY780 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, rad52δ::kanr KMY821 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, rad52δ::kanr KMY1596 KMY1613 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr, rad52δ::hphr MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr, rad52δ::hphr KMY1225 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, dnl4δ::kanr KMY1240 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, dnl4δ::kanr KMY1650 KMY1666 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr, dnl4δ::hphr MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, lrs4δ::kanr, dnl4δ::hphr KMY856 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, ku70δ::kanr KMY1083 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, ku70δ::kanr KMY2148 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, pol32δ::hphr KMY2160 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, pol32δ::hphr KMY2151 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, dpb3δ::hphr KMY2156 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, dpb3δ::hphr KMY875 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, sae2δ::hphr KMY1130 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, sae2δ::hphr KMY862 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, sir4δ::hphr KMY1095 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, sir4δ::hphr KMY852 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, sir3δ::kanr KMY1089 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, sir3δ::kanr KMY1148 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, sir3δ::kanr rad52δ::hphr KMY1178 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, sir3δ::kanr, S9

rad52δ::hphr KMY576 1 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs KMY643 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs KMY783 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, rad52δ::kanr KMY824 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, rad52δ::kanr KMY859 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, ku70δ::kanr KMY1086 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, ku70δ::kanr KMY1119 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, dnl4δ::kanr KMY1136 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, dnl4δ::kanr KMY779 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, nup84δ::kanr KMY846 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, nup84δ::kanr KMY733 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, esc1δ::hphr KMY760 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, esc1δ::hphr KMY691 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, lrs4δ::kanr KMY714 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, lrs4δ::kanr KMY694 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, csm1δ::kanr KMY718 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, csm1δ::kanr KMY697 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, heh1δ::kanr KMY722 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, heh1δ::kanr KMY699 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, nur1δ::kanr KMY726 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, nur1δ::kanr KMY1595 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, cik1δ::hphr KMY1626 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, cik1δ::hphr KMY865 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, sir4δ::hphr KMY1098 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, sir4δ::hphr KMY854 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, sir3δ::kanr KMY1092 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, sir3δ::kanr KMY1156 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, slx8δ::hphr KMY1184 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, slx8δ::hphr KMY1160 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, slx5δ::hphr KMY1190 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, slx5δ::hphr KMY1159 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, slx8δ::hphr KMY1187 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, slx8δ::hphr KMY1163 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, slx5δ::hphr KMY1193 MATa, ura3-δ0, leu2δ1, his3δ200, lys2δ202, ykl201c::csura3cs, slx5δ::hphr KMY1171 S10

KMY1205 KMY1166 KMY1199 KMY1169 KMY1202 KMY1593 KMY1607 KMY1594 KMY1647 KMY578 1 KMY1683 KMY1684 KMY1780 KMY2011 KMY580 1 KMY2189 KanR KanR MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Lrs4-Myc13- KanR MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Lrs4-Myc13- KanR MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Sir3-Myc13- KanR MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Sir3-Myc13- KanR KanR, lrs4δ::hphr KanR, lrs4δ::hphr KanR, cik1δ::hphr KanR, cik1δ::hphr MATa, ura3δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3cs::tet0112::natmx, LEU2::tetR-GFP MATa, ura3δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3cs::tet0112::natmx, LEU2::tetR-GFP, Nup49-GFP-KanR MATa, ura3δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3cs::tet0112::natmx, LEU2::tetR-GFP, Nup49-GFP-KanR, lrs4δ::hphr MATa, ura3δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3cs::tet0112::natmx, LEU2::tetR-GFP, Nup49-GFP-KanR, nur1δ::hphr MATa, ura3δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3cs::tet0112::natmx, LEU2::tetR-GFP, Nup49-GFP-KanR, cik1δ::hphr MATa, ura3δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3cs::tet0112::natmx, LEU2::tetR-GFP, nup145cδ::kanmx4 MATa, ura3δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3cs::tet0112::natmx, LEU2::tetR-GFP, nup145cδ::kanmx4, Nup49-GFP-His3 KMY2190 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Cik1-Myc-NatR KMY2263 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Cik1-Myc-NatR KMY2193 KMY2266 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Lrs4Δ::KanR, Cik1-Myc-NatR MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Lrs4Δ::KanR, Cik1-Myc-NatR KMY219 Heh1-TAP-TRPkl, Lrs4-Myc13-KanR KMY1910 Heh1-TAP-TRPkl, Lrs4-Myc13-KanR KMY2058 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Nup84-Myc- NatR, Lrs4-TAP-KanR S11

KMY2074 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, Nup84-Myc- NatR, Lrs4-TAP-KanR KMY2250 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, rad9δ::natr KMY2275 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, rad9δ::natr KMY2253 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, swr1δ::natr KMY2278 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, swr1δ::natr KMY2256 KMY2281 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cik1δ::kanr, swr1δ::natr MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cik1δ::kanr, swr1δ::natr KMY2012 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, tub3δ::hphr KMY2027 MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, tub3δ::hphr KMY2018 KMY2033 KMY2258 KMY2289 KMY2261 KMY2292 KMY1738* KMY1861* KMY1733* KMY1849* KMY2076* KMY2078* KMY2009* KMY2067* KMY2050* MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cik1δ::kanr, tub3δ::hphr MATa, ura3-δ851, leu2δ1, his3δ200, lys2δ202, ykl222c::csura3csa, cik1δ::kanr, tub3δ::hphr KanR, swr1δ::natr KanR, swr1δ::natr KanR, cik1δ::hphr, swr1δ::natr KanR, cik1δ::hphr, swr1δ::natr NatR NatR NatR NatR NatR, Nup84-Myc13-KanR NatR, Nup84-Myc13-KanR NatR, Nup84-Myc13-KanR, lrs4δ::hphr NatR, Nup84-Myc13-KanR, lrs4δ::hphr NatR, Nup84-Myc13-KanR, cik1δ::hphr S12

KMY2070* KMY2036* KMY2061* KMY2051* KMY2064* KMY2057* KMY2071* KMY1815* KMY1864* KMY1800* KMY1852* KMY1847* KMY1867* KMY1803* KMY1855* KMY1913* KMY1989* KMY1807* KMY1858* DDY3393 2 NatR, Nup84-Myc13-KanR, cik1δ::hphr NatR, Nup84-Myc13-KanR NatR, Nup84-Myc13-KanR NatR, Nup84-Myc13-KanR, lrs4δ::hphr NatR, Nup84-Myc13-KanR, lrs4δ::hphr NatR, Nup84-Myc13-KanR, cik1δ::hphr NatR, Nup84-Myc13-KanR, cik1δ::hphr NatR, lrs4δ::hphr NatR, lrs4δ::hphr NatR, lrs4δ::hphr NatR, lrs4δ::hphr NatR, cik1δ::hphr NatR, cik1δ::hphr NatR, cik1δ::hphr NatR, cik1δ::hphr NatR, nup84δ::hphr NatR, nup84δ::hphr NatR, nup84δ::hphr NatR, nup84δ::hphr JRL346 Mata::HOcsDEL::hisG ura3 DEL851 trp1del63 sup53del::leu2del::natmx hmldel::hisg hmrdel::ade3 ade3::gal10::ho can1,1-1446::hocs::hph::del AVT2 ykl215c::leu2::hisg::can1del1-289 DDY3398 JRL346 pol32::kanmx DDY3394 JRL346 cik1::kanmx S13

DDY3396 JRL346 kar3::kanmx DDY4009 JRL346 lrs4::kanmx (clone #1) DDY4010 JRL346 lrs4::kanmx (clone #2) DDY4011 JRL346 nup84::kanmx (clone #1) DDY4012 JRL346 nup84::kanmx (clone #2) DDY4013 JRL346 kar3::kanmx prs414 DDY4014 JRL346 kar3::kanmx prs414-kar3 DDY4015 JRL346 kar3::kanmx prs414-kar3-1 DDY4016 JRL346 kar3::kanmx prs414-kar3-898 DDY2210 3 DDY4008 DDY3094 DDY3095 DDY3093 DDY3096 DDY355 8 DDY3091 DDY3089 DDY3090 DDY3092 DDY2932 4 DDY3430 DDY3431 DDY2973 5 DDY3099 YMV45 ho hml::ade1 mata::hisg hmr::ade1 leu2::leu2(asp718-sali)-ura3-pbr332- MATa ade3::gal::ho ade1 lys5 ura3-52 trp1::hisg YMV45 ho hml::ade1 mata::hisg hmr::ade1 leu2::leu2(asp718-sali)-ura3-pbr332- MATa ade3::gal::ho ade1 lys5 ura3-52 trp1::hisg rad52::kanmx YMV45 ho hml::ade1 mata::hisg hmr::ade1 leu2::leu2(asp718-sali)-ura3-pbr332- MATa ade3::gal::ho ade1 lys5 ura3-52 trp1::hisg cik1::kanmx YMV45 ho hml::ade1 mata::hisg hmr::ade1 leu2::leu2(asp718-sali)-ura3-pbr332- MATa ade3::gal::ho ade1 lys5 ura3-52 trp1::hisg kar3::kanmx YMV45 ho hml::ade1 mata::hisg hmr::ade1 leu2::leu2(asp718-sali)-ura3-pbr332- MATa ade3::gal::ho ade1 lys5 ura3-52 trp1::hisg nup84::kanmx YMV45 ho hml::ade1 mata::hisg hmr::ade1 leu2::leu2(asp718-sali)-ura3-pbr332- MATa ade3::gal::ho ade1 lys5 ura3-52 trp1::hisg htz1::kanmx YMV2 MATD::hisG hml::ade1 hmr::ade1 his4::ura3::trp1-leu2 (Xho1-toAsp718)- pbr322-his4 ade1 lys5 ura3-52 trp1::hisg leu2::hocs ade3::gal::ho YMV2 MATD::hisG hml::ade1 hmr::ade1 his4::ura3::trp1-leu2 (Xho1-toAsp718)- pbr322-his4 ade1 lys5 ura3-52 trp1::hisg leu2::hocs ade3::gal::ho rad52::natmx YMV2 MATD::hisG hml::ade1 hmr::ade1 his4::ura3::trp1-leu2 (Xho1-toAsp718)- pbr322-his4 ade1 lys5 ura3-52 trp1::hisg leu2::hocs ade3::gal::ho cik1::natmx YMV2 MATD::hisG hml::ade1 hmr::ade1 his4::ura3::trp1-leu2 (Xho1-toAsp718)- pbr322-his4 ade1 lys5 ura3-52 trp1::hisg leu2::hocs ade3::gal::ho kar3::natmx YMV2 MATD::hisG hml::ade1 hmr::ade1 his4::ura3::trp1-leu2 (Xho1-toAsp718)- pbr322-his4 ade1 lys5 ura3-52 trp1::hisg leu2::hocs ade3::gal::ho nup84::kanmx YMV2 MATD::hisG hml::ade1 hmr::ade1 his4::ura3::trp1-leu2 (Xho1-toAsp718)- pbr322-his4 ade1 lys5 ura3-52 trp1::hisg leu2::hocs ade3::gal::ho htz1::kanmx BY4743 Mata/α his3δ1/his3δ1 leu2δ0/leu2δ0 lys2δ0/lys2 met15δ0/met15 ura3δ 0/ura3 est2::kanmx/est2 cik1::natmx BY4743 Mata/α his3δ1/his3δ1 leu2δ0/leu2δ0 lys2δ0/lys2 met15δ0/met15 ura3δ 0/ura3 est2::kanmx/est2 kar3::natmx TGI354 hoδ MATa-inc arg5,6::mata-hph ade3::gal-ho hmrδ::ade hmlδ ::ADE1 ura3-52 TGI354 hoδ MATa-inc arg5,6::mata-hph ade3::gal-ho hmrδ::ade hmlδ ::ADE1 ura3-52 cik1::natmx DDY3100 TGI354 hoδ MATa-inc arg5,6::mata-hph ade3::gal-ho hmrδ::ade hmlδ ::ADE1 S14

ura3-52 rad52::natmx DDY4302 SubTEL Nup49-mCherry::HPH prs413-rad52-yfp DDY4303 SubTEL cik1::kanmx Nup49-mCherry::HPH prs413-rad52-yfp DDY4296 JRL346 Nup49-mCherry::URA3 prs415-rad52-yfp DDY4297 JRL346 Nup49-mCherry::URA3 prs415-rad52-yfp cik1::kanmx DDY4298 JRL346 Nup49-mCherry::URA3 prs415-rad52-yfp kar3::kanmx DDY4299 JRL346 Nup49-mCherry::URA3 prs415-rad52-yfp kar3::kanmx prs414-kar3 DDY4300 JRL346 Nup49-mCherry::URA3 prs415-rad52-yfp kar3::kanmx prs414-kar3-1 DDY4301 JRL346 Nup49-mCherry::URA3 prs415-rad52-yfp kar3::kanmx prs414-empty *cmrs-dsb-l = Control sequence, MRS-DSB-L = zip sequence S15

Supplementary Table 4. List of primer pairs used for PCR survivor analysis, ChIP analysis, and gemic integration of NPC-targeting DNA zip/control sequences. Location (Application) Across SubTEL DSB (Survivor) Across SubTEL DSB (zip/cont. survivor) Telomere (Generic AC 1-3 sequence) 0.7 kb left of SubTEL DSB (ChIP) 0.7 kb right of SubTEL DSB (ChIP) 0.07 kb to TEL VI R (ChIP) Control locus ACT1 (ChIP) Amplifying product for transformation (zip integration) Amplifying product for transformation (Control integration) 0.6 kb left of SubTEL DSB (zip/cont. ChIP) 1.5 kb away from n-telomeric DSB (ChIP) Size (bp) 1966 664 (NHEJ) 1798 496 (NHEJ) Sequence 1 Sequence 2 CTGAGTCTGCACTAGACAAT (P1) GAAGTTAAGTGCGCAGAAAG (P1*) ATCTTGATCTCAAAAGCACC (P2) ATCTTGATCTCAAAAGCACC (P2) variable ACCACACACCCACCAC (P3) ATCTTGATCTCAAAAGCACC (P2) 200 TTCTCTCAATACCATCCGC AGAAGCTTATTGTCTAAGCCC 350 GGGAGTAAATTAGACTATGC TAGAATACTTTGCATTCCCA 270 CATGACCAGTCCTCATTTCCATC ACGTTTAGCTGAGTTTAACGGTG 153 GCCTTCTACGTTTCCATCCA GGCCAAATCGATTCTCAAAA n/a n/a CCCAAAAAAAAGAGCAACTATCTAG AAAAGTATGTAGAAGTCCTTCTTTC CCGGATCCCCGGGTTAATTAA CCCAAAAAAAAGAGCAACTATCTAG AAAAGTATGTAGAAGTCGATCATT GCCGGATCCCCGGGTTAATTAA TTTGTGAAAGCTATTGGTGGCACC CTCTGTTCCTTTCGGAGAATTCGAG CTCGTTTAAAC TTTGTGAAAGCTATTGGTGGCACC CTCTGTTCCTTTCGGAGAATTCGAG CTCGTTTAAAC 362 TTCTCACATCACATCCGAACA ATGTCCTCGACGGTCAGC 268 (qpcr only) GTATGACTCACCCGGAAACC ATAACGGCAAACAGCAAAGG S16

Supplementary References 1 Therizols, P. et al. Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region. J. Cell Biol. 172, 189-199 (2006). 2 Lydeard, J. R., Lipkin-Moore, Z., Jain, S., Eapen, V. V. & Haber, J. E. Sgs1 and exo1 redundantly inhibit break-induced replication and de vo telomere addition at broken chromosome ends. PLoS Genet 6, e1000973 (2010). 3 Vaze, M. B. et al. Recovery from checkpoint-mediated arrest after repair of a doublestrand break requires Srs2 helicase. Mol Cell 10, 373-385 (2002). 4 Yeung, M. & Durocher, D. Srs2 enables checkpoint recovery by promoting disassembly of DNA damage foci from chromatin. DNA Repair (Amst) 10, 1213-1222 (2011). 5 Ira, G., Malkova, A., Liberi, G., Foiani, M. & Haber, J. E. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115, 401-411 (2003). S17