Journal:

Article Title: The Yeast Chromatin Remodeler RSC Complex Facilitates End Joining Repair of DNA Double-Strand Breaks †

doi: 10.1128/MCB.25.10.3934-3944.2005

Figure Lengend Snippet: Interaction of Rsc1 and Rsc2 with yKu80 and Mre11. (A) A series of yeast two-hybrid assays were carried out to examine interactions of Rsc1, the rsc1-1M and rsc1-2M mutants, and Rsc2 with several NHEJ factors. Interactions were discerned based on the ability to express the HIS3 reporter gene by spotting cells on synthetic medium in the presence or absence of histidine. (B) Lysates from yeast cells coexpressing GST-Mre11 or GST and Rsc1-TAP, Rsc2-TAP, Rsc4-TAP, or Swi3-TAP were incubated with glutathione-Sepharose 4B, and the TAP-tagged proteins associated with GST-Mre11 were detected by immunoblotting with peroxidase-antiperoxidase (PAP). GST-Mre11 was detected by anti-Mre11 antibody (α-Mre11). The 1/10 volume of the yeast extracts used for each pull-down experiment is shown as input. (C) 35S-labeled Rsc1, rsc1-1M and rsc1-2M mutant proteins, and Rsc2, obtained by coupled in vitro transcription-translation, were incubated with GST-Mre11 or GST. Proteins associated with GST-Mre11 or GST were isolated on glutathione-Sepharose beads, resolved by SDS-PAGE, and visualized by autoradiography. The numbers below lanes 2, 4, 6, and 8 report the relative affinities of the 35S-labeled proteins for GST-Mre11, which were calculated by comparing the signal intensities of the Rsc1 or the rsc1 mutant derivatives pulled down from the identical amount of GST-Mre11, normalized by the amount of Rsc1 or rsc1 mutants before pull down.

Article Snippet: The plasmids pGAD-T7- RSC1 , pGAD-T7- RSC1- 1M, pGAD-T7-RSC1-2M, and pGAD-T7- RSC2 were used as templates to synthesize 35 S-labeled Rsc1, rsc1-1M, rsc1-2M, and Rsc2 by TNT-coupled reticulocyte lysate systems (Promega).

Techniques: Incubation, Western Blot, Labeling, Mutagenesis, In Vitro, Isolation, SDS Page, Autoradiography

Journal:

Article Title: The Yeast Chromatin Remodeler RSC Complex Facilitates End Joining Repair of DNA Double-Strand Breaks †

doi: 10.1128/MCB.25.10.3934-3944.2005

Figure Lengend Snippet: Attenuated interactions between Rsc1 and Mre11 sensitized cells to genotoxic stresses. (A) Alignment of Rsc1 bromodomains with the bromodomains of yeast Gcn5, P/CAF, Rsc2, and Rsc4. Regions of identity among the five bromodomains are highlighted in gray. Four alpha-helix bundles and two long loop structures predicted by nuclear magnetic resonance studies are shown (19). Locations of the mutations in Rsc1 that reduced the interaction with Mre11 without affecting binding to yKu80 are marked by arrows. (B) Yeast extracts (200 μg) with a centromeric plasmid expressing FLAG-tagged RSC1, rsc1-1M, or rsc1-2M were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were separated on SDS-PAGE, transferred to polyvinylidene difluoride membrane, and then probed with anti-Sth1 antiserum (a gift from B. Laurent). The 1/10 volume of extract used for immunoblotting with anti-Sth1 antibody is shown as input. The level of Rsc1 or rsc1 mutant protein expression was analyzed by Western blot analysis of whole-cell extracts using anti-FLAG antiserum (Sigma). (C) Yeast strains with RSC1 deleted but harboring a plasmid that expresses either wild-type RSC1, rsc1-1M, or rsc1-2M were generated by standard yeast transformation procedures. Serial dilutions of mid-log-phase cultures were plated onto YEPD medium with or without phleomycin. Plates were incubated at 30°C for 3 to 4 days before they were photographed.

Article Snippet: The plasmids pGAD-T7- RSC1 , pGAD-T7- RSC1- 1M, pGAD-T7-RSC1-2M, and pGAD-T7- RSC2 were used as templates to synthesize 35 S-labeled Rsc1, rsc1-1M, rsc1-2M, and Rsc2 by TNT-coupled reticulocyte lysate systems (Promega).

Techniques: Nuclear Magnetic Resonance, Binding Assay, Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page, Western Blot, Mutagenesis, Generated, Transformation Assay, Incubation