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. 2024 Dec 4;13:RP96933. doi: 10.7554/eLife.96933

Figure 1. Y2H screens suggest interaction between ScRev7 and the MRX subunits.

The Y2H assay was performed in (A) wild-type, (B) rev3Δ, and (C) mre11Δ rad50Δ xrs2Δ mutant strains in PJ69-4A background. These strains were co-transformed with pairwise combinations of empty vector, bait (pGBKT7-REV7) and prey (pGADT7/MRE11, RAD50, XRS2, REV7, or SAE2) plasmids. Equal number of mid-log phase cells was spotted onto the SC/-Trp-Leu agar plates (upper panels) or SC/-Trp -Leu-His agar plates containing 3-aminotriazole (3-AT) (bottom panels). Cells were imaged after 48 hr of growth at 30°C. The images shown in panels (A–C) are representative of three independent experiments. (D) Quantitative parameters for interaction between ScRev7 and Rev1, Mre11, Rad50, Xrs2, or Mre11–Rad50 proteins.

Figure 1.

Figure 1—figure supplement 1. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of purified proteins used in this study.

Figure 1—figure supplement 1.

(A) SDS–PAGE and western blot analysis of purified GFP-tagged ScRev7 and eGFP. Lane 1 (in panels A–F) standard molecular weight markers; lane 2, purified GFP-tagged ScRev7; lane 3, purified GFP. Western blot analysis of GFP-tagged Rev7 (lane 4) and eGFP (lane 5) using mouse anti-GFP antibodies. (B) Purified GFP-tagged Rev7-C1 (lane 2) and GFP-tagged Rev7 (lane 3). Western blot analysis of GFP-tagged Rev7-C1 (lane 4) and GFP-tagged Rev7 (lane 5) using mouse anti-GFP antibodies. (C) Purified MRX subunits: lane 2, Mre11; lane 3, Rad50; lane 4, Xrs2. (D) Purified GST-tagged Rev1; the faster migrating band (*) corresponds to ScRev7, which co-purifies with GST-tagged Rev1 (Acharya et al., 2006). (E) Purified Sae2 and (F) purified Dmc1.
Figure 1—figure supplement 1—source data 1. Original files for gel images and blots displayed in Figure 1—figure supplement 1.
Figure 1—figure supplement 1—source data 2. PDF file containing labelled uncropped gel images and blots displayed in Figure 1—figure supplement 1.
Figure 1—figure supplement 2. Mass spectromtery analysis of purified Rev7-GFP.

Figure 1—figure supplement 2.

Representative spectra for unique peptides obtained after trypsin digestion of Rev7-GFP protein that matches with, (A) GFP sequence and (B) ScRev7 sequence. (C) Snapshot of sequence coverage (shown in green) obtained for Rev7 and (D) GFP. (E) Table summarizes data obtained for Orbitrap Liquid chromatography mass-spectrometry performed using purified Rev7-GFP protein.
Figure 1—figure supplement 3. Microscale thermophoresis (MST) reveals a direct interaction between Rev7 and MRX subunits.

Figure 1—figure supplement 3.

Normalized MST-binding curves were generated for the titration of (A) Mre11, (B) Rad50, (C) Xrs2, (D) Rev1, (E) the Mre11–Rad50 complex, and (F) Sae2 proteins against Rev7-eGFP. Panel F also includes datasets for the binding of Mre11, Rad50, and Xrs2 against eGFP. Error bars represent the standard error of the mean (SEM), and data are representative of three independent experiments. Panel F represents data from two independent experiments.
Figure 1—figure supplement 3—source data 1. Raw data for panels A–F.
Figure 1—figure supplement 4. Rev7-C1 exhibits weak interactions with MRX subunits.

Figure 1—figure supplement 4.

Binding isotherms were obtained by incubating increasing concentrations of (A) Mre11 (0.00015–5 μM), (B) Rad50 (0.00006–2 μM), and (C) MR complex (0.00015–5 μM) with fixed concentration of Rev7C1:GFP protein (0.25 μM). (D) Kd values obtained upon fitting the curves in Kd model using MO affinity analysis software (Nanotemper). Data are representative of three independent experiments.
Figure 1—figure supplement 4—source data 1. Raw data for panels A–C.