ruby2 fluorophore addgene pfa6a link yomruby2 kan plasmid  (Addgene inc)

Journal: Cell reports

Article Title: Asymmetric Processing of DNA Ends at a Double-Strand Break Leads to Unconstrained Dynamics and Ectopic Translocation.

doi: 10.1016/j.celrep.2018.07.102

Figure Lengend Snippet: Figure 4. Break Ends at TG-Flanked DSBs Separate and Move in an Uncoordinated Fashion (A) System used to visualize Rad52-Ruby2 on one side of the resected HO-induced non-TG break side, while the TG side was visualized by LacI-GFP. Criteria for juxtaposition of foci are shown. Percentage of colocalization of LacI-GFP and Rad52-Ruby2 foci in Tg0 (GA-9948) and Tg80 (GA-9912) are quantified at 135 min after HO induction. Cleavage efficiency is >95%. n = 80 cells per strain per experiment; mean values of three independent experiments ± SEM are shown. (B) Scheme of LacI-GFP and Rad52-Ruby2 locus tracking by TIRF microscopy acquired at 80-ms time intervals for 1 min, starting 2 hr after HO induction. (C) MSD analysis based on SPTs of LacI-GFP and Rad52-Ruby2 using Tg0 (GA-9948, black) and Tg80 (GA-9913, orange) strains, with and without HO cut. SPTs per strain and conditions are as follows: Tg0 uncut, 20; Tg0 Rad52-Ruby2, 23; Tg80 uncut, 23; Tg0 LacI-GFP, 25; Tg80 Rad52-Ruby2, 24; and Tg80 LacI-GFP, 24. Rc, radii of constrained movement (mm), are indicated above each averaged track. (D) Statistical biophysical parameters (Amitai et al., 2017) determined from single-particle trajectories as in (C). Numbers are means of at least 20 trajectories. a, anomalous exponent; Dc, diffusion coefficient; kc, effective spring constant; Lc, length of constraint (Amitai et al., 2017). (E) In the absence of TG repeats, ends are held together by the MRX complex, which leads to end resection, RPA, Rad51, and Rad52 binding. In Tg80, MRX binds only the non-TG side. Ends separate, moving without constraint.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mab414 nuclear pore antibody abcam Mab414, ab24609 Sheep anti-mouse IgG magnetic beads Invitrogen 11031 Anti-HA antibody Santa Cruz Biotech F-7, sc-7392 Anti-PK antibody Acris Antibodies SV5-PK1, SM1691 Sheep anti-rabbit IgG Dynabeads Invitrogen 11203D anti-yKu70 rabbit polyclonal antibody A. E. Tomkinson anti-Hdf1 Critical Commercial Assays SMRTbell Template Prep Kit Pacific Biosciences 100-259-100 PippinHT Sage Science HTP0001 protease inhibitors (cOmplete EDTA-free) Roche 04693159001 AccuPrep DNA extraction kit Bioneer K-3034 MagBead-binding One Cell Per Well Pacific Biosciences 100-267-800-03 Binding Kit P6 v2 Pacific Biosciences 100-372-700 DNA Sequencing Kit 4.0 Pacific Biosciences 100-364-600 Deposited Data Pacific Biosciences sequencing datasets NCBI Bioproject database Submission ID SUB4312748, under Bioproject ID PRJNA482327 Experimental Models: Organisms/Strains Budding yeast: see Table S1 Susan Gasser, FMI Table S1 Oligonucleotides Primer lists: see Tables S2 and S3 different suppliers, this paper Tables S2 and S3 Recombinant DNA Plasmid expressing wild-type ULS1 HFP269, H. Ferreira, St. Andrews University p416-FLAG-ULS1 Plasmid expressing translocase mutant uls1-K975A HFP282, H. Ferreira, St. Andrews University p416-FLAG-uls1-K975A Plasmid containing EXO1 Lee et al., 2003 pJH1772 Plasmid containing Ruby2 fluorophore Addgene pFA6a-link-yomRuby2-Kan Plasmid containing TG80-HO-30MNT2 Ribeyre and Shore, 2012 pIM35 Plasmid containing (TTAGGG)60-HO30MNT2 Ribaud et al., 2012 pVR4 Plasmid 3571 containing TG250-HO30MNT2 This paper pUC57-TG250 Plasmid 3892 containing TG18-HO-30MNT2 This paper pUC57-TG18 Software and Algorithms SPT analysis and biophysical parameter extraction S. Gasser, Amitai et al., 2017 NA Spot tracker ImageJ (FIJI) plug-in S. Gasser, Sage et al., 2005 NA PointPicker S. Gasser, Meister et al., 2010 NA

Techniques: Microscopy, Single Particle, Diffusion-based Assay, Binding Assay

Journal: Cell reports

Article Title: Asymmetric Processing of DNA Ends at a Double-Strand Break Leads to Unconstrained Dynamics and Ectopic Translocation.

doi: 10.1016/j.celrep.2018.07.102

Figure Lengend Snippet: Figure 6. Siz2 and Uls1 Control Relocation of the TG Side at the Tg80 DSB and Suppress NHEJ (A) Scheme of major yeast SUMO-dependent ubiquitin ligases (STUbLs) and RNF4 in man. Uls1 contains a SNF2-like ATPase as well as SUMO-interacting motifs (SIMs) and RING finger ubiquitin ligase domain. (B) Zoning assay (Figure 1B) for DSB distribution at 130 min after galactose-induced HO expression. Strains used were Tg80 (GA-8119), Tg80 slx8D (GA-10050), Tg80 uls1D (GA-9855), and Tg80 siz2D (GA-9794). Mean values of three independent experiments ± SEM are shown. *Non-random distribution in zone 1 (c2 test of random versus experimental distribution; degree of freedom, 2; confidence limit, 95%), p = 0.048. (C) Co-localization of MAT with the pore cluster in nup133DN background, in wild-type (GA-7314), slx5D (GA-7969), uls1D (GA-8475), and siz2D (GA-7970) strains at specific times after HO induction. Pink and red colors indicate adjacency and colocalization, respectively (Horigome et al., 2016). Gray, random distribution zone based on theoretical tests. (D) Indicated genes were deleted in the Tg80 strain (wild-type, GA-8119) generating GA-10050, GA-9855, GA-9794, GA-9158, and GA-9449. Graph presents the percentage of colonies repaired by imprecise NHEJ out of all survivors on galactose, as scored by qPCR across the HO cut site (Figure 1C). n = 60 per strain. *Statistical significance with a p value < 104 in a c2 test of wild-type and mutant with 95% confidence interval. Example gels showing 20 colonies of indicated strains are shown. (E) Loss of Uls1 does not release the resection block on the TG side of the DSB in Tg80. Resection scored by ssDNA AluI assay (Figure 2A) with Tg80 uls1D (GA-9555). Probe distance from the HO consensus is shown. Three biological replicates, assayed in triplicate, are presented as mean values ± SEM. (F) MSD analysis based on single-particle trajectories of LacI-GFP and Rad52-Ruby2 in Tg80 (GA-9913, orange) and Tg80 uls1D (GA-10435, green), with and without HO induction (cut versus uncut). Tg80 control data are from Figure 4C. Videos analyzed per strain are as follows: Tg80 lacI-GFP uncut, 23; Tg80 Rad52- Ruby2, 28; Tg80 lacI-GFP cut, 28; Tg80 uls1D LacI-GFP uncut, 43; Tg80 uls1D Rad52-Ruby2 cut, 25; Tg80 uls1D LacI-GFP cut, 9. Rad52 foci in the Tg80 uls1D are rare due to elevated rates of NHEJ.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mab414 nuclear pore antibody abcam Mab414, ab24609 Sheep anti-mouse IgG magnetic beads Invitrogen 11031 Anti-HA antibody Santa Cruz Biotech F-7, sc-7392 Anti-PK antibody Acris Antibodies SV5-PK1, SM1691 Sheep anti-rabbit IgG Dynabeads Invitrogen 11203D anti-yKu70 rabbit polyclonal antibody A. E. Tomkinson anti-Hdf1 Critical Commercial Assays SMRTbell Template Prep Kit Pacific Biosciences 100-259-100 PippinHT Sage Science HTP0001 protease inhibitors (cOmplete EDTA-free) Roche 04693159001 AccuPrep DNA extraction kit Bioneer K-3034 MagBead-binding One Cell Per Well Pacific Biosciences 100-267-800-03 Binding Kit P6 v2 Pacific Biosciences 100-372-700 DNA Sequencing Kit 4.0 Pacific Biosciences 100-364-600 Deposited Data Pacific Biosciences sequencing datasets NCBI Bioproject database Submission ID SUB4312748, under Bioproject ID PRJNA482327 Experimental Models: Organisms/Strains Budding yeast: see Table S1 Susan Gasser, FMI Table S1 Oligonucleotides Primer lists: see Tables S2 and S3 different suppliers, this paper Tables S2 and S3 Recombinant DNA Plasmid expressing wild-type ULS1 HFP269, H. Ferreira, St. Andrews University p416-FLAG-ULS1 Plasmid expressing translocase mutant uls1-K975A HFP282, H. Ferreira, St. Andrews University p416-FLAG-uls1-K975A Plasmid containing EXO1 Lee et al., 2003 pJH1772 Plasmid containing Ruby2 fluorophore Addgene pFA6a-link-yomRuby2-Kan Plasmid containing TG80-HO-30MNT2 Ribeyre and Shore, 2012 pIM35 Plasmid containing (TTAGGG)60-HO30MNT2 Ribaud et al., 2012 pVR4 Plasmid 3571 containing TG250-HO30MNT2 This paper pUC57-TG250 Plasmid 3892 containing TG18-HO-30MNT2 This paper pUC57-TG18 Software and Algorithms SPT analysis and biophysical parameter extraction S. Gasser, Amitai et al., 2017 NA Spot tracker ImageJ (FIJI) plug-in S. Gasser, Sage et al., 2005 NA PointPicker S. Gasser, Meister et al., 2010 NA

Techniques: Control, Ubiquitin Proteomics, Expressing, Mutagenesis, Blocking Assay, Single Particle