Crucial roles for Ligase III in mitochondria and alt NHEJ but not in Xrcc1 dependent DNA repair Deniz Simsek Erika Brunet Amy Furda Sachin Katyal Yankun Gao Sunnie Yan Wai Wong J r me Artus Yannick Doyon Jacqueline Lou Lei Zhang James Li Edward J Rebar Philip D Gregory Michael C Holmes Anna Katerina Hadjantonakis Stewart Shuman Bennett Van Houten Peter J McKinnon Maria Jasin SEVEN SUPPLEMENTARY FIGURES Supplementary Figure 1 5 2 4 5 8 6 10 8 6 13 10 15 13 18 15 20 18 B Lig3 cDNA 20 MtLig3 cDNA M88T 2 4 5 8 6 10 13 15 NucLig3 cDNA 20 18 1 neo 2 KO 4 5 15 18 20 Cre expression generating second KO allele KO cKOneo Lig3 cDNA C Cre expression generating first KO allele 1 WT cKO cKO cKOneo Targeting WT allele 2 KO KO 4 Lig3 cDNA 3a KO cKOneo MtLig3 cDNA 3b KO cKOneo 4 3c KO cKOneo NucLig3 cDNA KO KO MtLig3 cDNA 4 cKO neo Int5 6Fw2 Int5 6Rv cKO cKOneo neo Int5 6Fw2 cKO Int5 6Rv neo bp KO 600 500 cK On 560 bp eo cK On eo cKOneo KO 2 Generating first KO allele cK On cK On eo 560 bp bp 1 000 600 500 cKOneo neo cKOneo KO cKO no product Int5 6Rv eo Int5 6Fw2 T cK O cKOneo WT WT cKOcKOneo cKO W no product 500 bp cK O D 1 Targeting WT allele cK On eo integrating cDNA 4 neo Int5 6 Int16 17 2 500bp bp 3 000 2 000 1 000 KO cKOneo 4 WWT T cKO 2 cK O WT cK O A Simsek et al Supplementary Figures Supplementary Figure 1 a Targeting strategy to generate conditional Lig3 knock out alleles in mouse ES cells b Schematic of human Lig3 cDNAs Lig3 contains both the wild type nuclear and mitochondrial translation initiation sites NucLig3 lacks the mitochondrial leader sequence MLS and MtLig3 contains the MLS but is mutated at the nuclear translation initiation site M88T c Pre emptive complementation strategy for the generation of Lig3KO KO cells Steps 1 and 2 were used to generate the Lig3KO cKOneo parental cells for this study Step 3 is the stable random integration of Lig3 cDNAs to generate the transgenic cell lines using a cotransfected hygromycin resistance gene Step 4 is Cre expression to determine if G418S that is Lig3KO KO clones could be generated from the transgenic lines d PCR analysis to confirm the expected genotypes in the generation of the Lig3KO cKOneo parental cells Supplementary Figure 2 A Mitotracker GFP Hoechst Overlay no cDNA D Lig3 GFP NucLig3 GFP MtLig3 GFP 20 m MtLig3 BRCT GFP B MtLig3 BRCT GFP NES MtLig3 BRCT GFP NES 20 m NucLig3 GFP NES C MtLig1 GFP MtLig1 NLS GFP Lig1 GFP 20 m Supplementary Figure 2 Live cell imaging to examine subcellular localization of transiently expressed GFP tagged Lig3 forms Plasmids expressing the various GFP tagged DNA ligases were transfected into mouse ES cells and imaged to visualize localization of the protein Note that this overexpression is sensitive for visualizing the protein but the levels of protein are much higher than in cells stably transfected with the ligase transgenes Tg used for experiments Mitochondria and the nucleus were labelled with Mitotracker Red CMXRos Invitrogen and Hoechst 33342 Invitrogen respectively a Wild type Lig3 GFP is found in the nucleus and mitochondria Nuclear localization of MtLig3 is reduced relative to Lig3 or NucLig3 due to mutation of the nuclear translation initiation site M88T it is likely not completely absent from the nucleus because it can still bind XRCC1 which has a nuclear localization signal b A potent nuclear export signal NES derived from MAPKK results in efficient export of even NucLig3 from the nucleus c Deletion of the NLS substantially reduces MtLig1 in the nucleus although a few cells still have residual nuclear expression Simsek et Supplementary Figures Supplementary Figure 3 RT PCR Western blotting NucLig3 cDNA pCAGGS backbone Hyg resistant clones Stable NucLig3 candidate clones bp 427 bp pCAGRTfw 1 000 hLig3Rv Lig3 100 400 cl7 Lig3 or MtLig3 cDNA cl8 cl13 cl20 tubulin 55 Hyg resistant clones bp bp 1 000 1 514 bp hLig3Rv pCAGRTfw 650 400 400 Flow cytometry for GFP fusion clones 100 100 100 80 80 80 60 60 60 of Max 40 of Max 40 of Max 40 20 20 20 0 0 0 10 0 10 1 10 2 FL1 H 10 3 10 4 10 0 10 1 10 2 FL1 H 10 3 10 4 10 0 10 1 10 2 FL1 H 10 3 Lig3KO cKOneo Lig3KO cKOneo Lig3 GFP cl4 Tg Supplementary Figure 3 Identification of clones expressing Lig3 transgenes The FL1 H indicated RT PCR primers are specific to the human Lig3 cDNA The forward primer binds to the pCAGGS expression vector and reverse primer at exon 3 of human Lig3 NucLig3 expression gives different sized products than Lig3 and MtLig3 RT PCR positive clones are tested for expression of Lig3 protein by Western blotting For GFPtagged clones flow cytometry detects a GFP population 10 4 Lig3 Lig3KO cKOneo MtLig3 GFP cl3 Tg KO cKOneo FL1 H Lig3KO cKOneo Lig3KO cKOneo NucLig3 GFP cl3 Tg of Max Lig3KO cKOneo kDA Supplementary Figure 4 G418s total no transgene total 39 17 41 24 24 145 0 0 0 0 0 0 0 0 0 0 0 0 Lig3 cl2 cl23 total 36 44 80 12 33 33 18 40 91 30 37 5 Lig3 GFP cl4 cl11 total 48 59 107 14 29 17 22 37 29 36 33 64 Lig3 delta ZNF GFP cl4 cl7 total 60 36 96 25 41 67 12 33 33 37 38 54 Lig3 delta BRCT GFP cl3 cl11 total 46 43 89 20 43 48 15 34 88 35 39 33 NucLig3 cl7 cl10 cl13 cl20 cl21 cl8 total 84 72 48 43 39 61 347 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NucLig3 GFP cl2 cl3 cl21 total 81 30 52 163 0 0 0 0 0 0 0 0 Supplementary Figure 4 con t G418s total MtLig3 cl5 cl10 cl11 cl20 total 48 77 60 54 239 33 39 25 22 119 68 75 50 65 41 67 40 74 49 79 MtLig3 GFP cl3 cl4 cl15 cl18 total 24 82 42 103 251 8 30 20 37 95 33 33 36 59 47 62 35 92 37 85 MtLig3 K508V cl2 cl12 total 82 70 152 0 0 0 0 0 0 MtLig3 K508V GFP cl9 cl14 total 46 127 173 0 0 0 0 0 0 MtLig3 delta ZNF GFP cl4 cl12 total 68 48 116 24 35 29 19 39 58 43 37 07 MtLig3 delta BRCT cl1 cl6 cl10 cl22 total 48 48 48 48 192 18 26 29 22 95 MtLig3 delta BRCT GFP cl15 cl18 cl24 total 72 54 30 156 30 41 67 17 31 48 18 60 65 41 67 MtLig3 delta BRCT NES GFP cl8 cl12 total 78 32 110 29 37 18 13 40 63 42 38 18 37 5 54 17 60 …
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