DOC PREVIEW
Berkeley MCELLBI 140 - Crucial roles for Ligase III in mitochondria

This preview shows page 1-2-20-21 out of 21 pages.

Save
View full document
View full document
Premium Document
Do you want full access? Go Premium and unlock all 21 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 21 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 21 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 21 pages.
Access to all documents
Download any document
Ad free experience
Premium Document
Do you want full access? Go Premium and unlock all 21 pages.
Access to all documents
Download any document
Ad free experience

Unformatted text preview:

SuppcoverSupp 1SuppFig1Supp2SuppFig2Supp3Supp4-1Supp4-2Supp4-3Supp5SuppFig5Supp6ASupp6BSupp6B-2SuppFig6Supp7-ASupp7E-1Supp7E-2Supp7E-3Supp7E-4SuppFig7Crucial 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, Jérôme 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 FIGURES5 15102181348206neo52 4 6 1510 18138202 4 5 6810 13 15 18 202 4 5 15 18 20WTcKOneo+cKOKO KO/cKOneo+WT/cKO cKO/cKOneo+ MtLig3 cDNANucLig3 cDNA123a3c44KO/KO4Targeting WT alleleCre expression: generating first KO alleleintegrating cDNACre expression: generating second KO allele3bLig3 cDNA4KO/KO Lig3 cDNAMtLig3 cDNA M88TNucLig3 cDNA1ABCSupplementary Figure 1cKOWTneocKOneo+D1. Targeting WT alleleInt5-6Fw2 Int5-6RvcKOInt5-6Fw2 Int5-6Rv560 bp500 bpno product500 600 1,000 WT WT/cKO cKOneo+/cKO 2. Generating first KO alleleneocKOneo+cKOKOneocKOneo+Int5-6Fw2 Int5-6Rv560 bp 500 600 neocKOneo+cKOKOneocKOneo+ 2,500bpInt5-6 Int16-17no product 1,000 3,000 2,000 bpbpbpWTWT/cKOcKO/cKOneo+KO/cKOneo+KO/cKOneo+KO/cKOneo+KO/cKOneo+Lig3 cDNA MtLig3 cDNAcKO/cKOneo+KO/cKOneo+cKO/cKOneo+Simsek et al, Supplementary FiguresSupplementary 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.DMitotracker OverlayHoechstGFPSupplementary Figure 2Lig3 GFPMtLig3 GFPNucLig3 GFPAMtLig3 ∆BRCTGFP 20µm20µmNucLig3GFP NESMtLig3 ∆BRCT GFP NESno cDNAMtLig3 ∆BRCT GFP NESMtLig1 GFPLig1 GFP 20µmBMtLig1 ∆NLS GFPCSupplementary 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.Supplementary Figure 3NucLig3 cDNApCAGGS backboneRT-PCR427 bp400 1,000 400 bpbpbpLig3 or MtLig3 cDNA1514 bp1,000 400 650 Flow cytometry for GFP fusion clones Western blotting α−Lig3α−tubulin100kDA55Lig3KO/ cKOneo+Stable NucLig3 candidate clones Hyg resistant clones Hyg resistant clones cl7 cl8 cl13 cl20100101102103104FL1-H020406080100% of Max% of MaxFL1-HLig3KO/ cKOneo+ Lig3KO/ cKOneo+ ; NucLig3 GFP cl3 TgLig3KO/ cKOneo+ Lig3KO/ cKOneo+ ; Lig3 GFP cl4 TgLig3KO/ cKOneo+ Lig3KO/ cKOneo+ ; MtLig3 GFP cl3 Tg100101102103104FL1-H020406080100% of Max100101102103104FL1-H020406080100% of MaxFL1-HpCAGRTfwpCAGRTfwhLig3RvhLig3Rv Supplementary Figure 3. Identification of clones expressing Lig3 transgenes. The 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 GFP-tagged clones, flow cytometry detects a GFP+ population. Simsek et, Supplementary Figurestotal#G418s%no transgene 39 0 017 0 041 0 024 0 024 0 0total: 145 0 0Lig3 cl2 36 12 33.33cl23 44 18 40.91total: 80 30 37.5Lig3 GFP cl4 48 14 29.17cl11 59 22 37.29total: 107 36 33.64Lig3 delta ZNF GFP cl4 60 25 41.67cl7 36 12 33.33total: 96 37 38.54Lig3 delta BRCT GFP cl3 46 20 43.48cl11 43 15 34.88total: 89 35 39.33NucLig3 cl7 84 0 0cl10 72 0 0cl13 48 0 0cl20 43 0 0cl21 39 0 0cl8 61 0 0total: 347 0 0NucLig3 GFP cl2 81 0 0cl3 30 0 0cl21 52 0 0total: 163 0 0Supplementary Figure 4total#G418s%MtLig3 cl5 48 33 68.75cl10 77 39 50.65cl11 60 25 41.67cl20 54 22 40.74total: 239 119 49.79MtLig3 GFP cl3 24 8 33.33cl4 82 30 36.59cl15 42 20 47.62cl18 103 37 35.92total: 251 95 37.85MtLig3 K508V cl2 82 0 0cl12 70 0 0total: 152 0 0MtLig3 K508V GFP cl9 46 0 0cl14 127 0 0total: 173 0 0MtLig3 delta ZNF GFP cl4 68 24 35.29cl12 48 19 39.58total: 116 43 37.07MtLig3 delta BRCT cl1 48 18 37.5cl6 48 26 54.17cl10 48 29 60.42cl22 48 22 45.83total: 192 95 49.48MtLig3 delta BRCT GFP cl15 72 30 41.67cl18 54 17 31.48cl24 30 18 60total: 156 65 41.67MtLig3 delta BRCT NES GFP cl8 78 29 37.18cl12 32 13 40.63total: 110 42 38.18Supplementary Figure 4, con’ttotal#G418s%Lig1 GFP cl10 48 0 0cl11 62 0 0total: 110 0 0MitLig1 GFP cl3 96 32 33.33cl16 78 29 37.18total: 174 61 35.06MitLig1 delta NLS GFP cl3 96 42 43.75cl16 34 9 26.47total: 130 51 39.23chlorella cl9 58 18 31.03cl20 44 23 52.27total: 102 41 40.2Mit chlorella cl3 40 13 32.5cl9 38 21 55.26total: 78 34 43.59Supplementary Figure 4, con’t Supplementary Figure 4.Summary of individual Cre experiments compiled in Figs. 1b and 2c. cl, Lig3 expressing cell line


View Full Document

Berkeley MCELLBI 140 - Crucial roles for Ligase III in mitochondria

Documents in this Course
CLINE 5

CLINE 5

19 pages

Prions

Prions

7 pages

Cline 10

Cline 10

15 pages

Cancer

Cancer

18 pages

CLINE 11

CLINE 11

19 pages

Cancer

Cancer

71 pages

Notes

Notes

12 pages

Midterm

Midterm

7 pages

The Gene

The Gene

17 pages

Two loci

Two loci

77 pages

Load more
Download Crucial roles for Ligase III in mitochondria
Our administrator received your request to download this document. We will send you the file to your email shortly.
Loading Unlocking...
Login

Join to view Crucial roles for Ligase III in mitochondria and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Crucial roles for Ligase III in mitochondria 2 2 and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?