Fig 1: The rad50-N18S mutation suppresses the DNA damage sensitivity of sae2? cells and decreases MRX abundance at DSBs(A and B) Exponentially growing cultures were serially diluted (1:10), and each dilution was spotted out onto YEPD plates with or without CPT or MMS.(C) ChIP and qPCR. Exponentially growing YEPR cell cultures were transferred to YEPRG to induce HO expression, followed by ChIP analysis of the recruitment of Mre11-Myc at the indicated distance from the HO-cut. In all diagrams, ChIP signals were normalized for each time point to the corresponding input signal. The mean values of three independent experiments are represented with error bars denoting SD. *p < 0.005 (Student’s t test).
Fig 2: Rif2 inhibits the endonuclease activity of Mre11-Rad50 (MR), but Mre11-Rad50-N18S (MRN18S) escapes this inhibition(A) Quantitation of ATPase assays was carried out with wild-type MR or MRN18S (both 100 nM) and Rif2 (500 nM), as indicated. Averages are shown from 5 independent experiments; error bars, SEM.(B) Scheme of the assay used to analyze the effect of Rif2 on the endonuclease activity of MR (which requires phosphorylated Sae2 [pSae2]), and the exonuclease activity of MR. 50-bp-long dsDNA, blocked with streptavidin on one end, was used as substrate.(C) Representative nuclease assays with MR, pSae2, and Rif2, as indicated.(D) Quantitation of endonuclease activity from experiments such as shown in (C) and (G). Averages are shown from 4 independent experiments; error bars, SEM.(E) Quantitation of exonuclease activity from experiments such as shown in (C) and (G). Averages are shown from 4 independent experiments; error bars, SEM.(F) MR and MRN18S used in this study.(G) Representative assays as in (C) but with MRN18S.
Fig 3: The function of Sae2 phosphorylation in the control of DNA end resection. Phosphorylation allows the formation of a tetrameric Sae2 form, which is capable of promoting the nuclease activity of Mre11-Rad50-Xrs2 (MRX). Additionally, phosphorylation of the C-terminal part of Sae2 is prerequisite for interaction with Rad50, which is also essential to promote MRX. These phosphorylation-dependent transitions mediate cell cycle and DNA-damage-dependent control of DNA end resection capacity of MRX. This prevents unscheduled DNA degradation by Mre11, aberrant recombination and resulting genome instability
Fig 4: Foci formation and DNA repair require MRX oligomerization in vivo.a Serial dilution of exponentially growing S. cerevisiae rad50\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\triangle$$\end{document}? cells expressing Rad50wt/ho/lo-yEVenus, spotted on agar plates (YPD) +/- DNA damage-inducing camptothecin (CPT) or hydroxyurea (HU), and incubated for 2 days at 30 °C. n = 3. b Doubling times of exponentially growing rad50\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\triangle$$\end{document}? cells and rad50\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\triangle$$\end{document}? strains expressing Rad50wt/ho/lo-yEVenus +/- CPT treatment. Mean and minimum to maximum values shown, n = 3 independent experiments. c Anti-Rad50 western blot showing galactose-induced overexpression of untagged Rad50wt/ho/lo in wild-type cells (+Gal-OE). Center panel is contrast-enhanced to show endogenous Rad50wt levels (wild-type, -Gal-OE). a-Tubulin controls equal loading. n = 3. d Serial dilution of exponentially growing wild-type cells expressing galactose-inducible Rad50wt/ho/lo, spotted on SD-URA agar plates with glucose (-) or galactose (+ variant overexpression), and incubated at 30 °C for 2 days. Before overexpression, cells carrying Rad50wt/ho/lo were similarly resistant to DNA damage as wild-type cells due to endogenous Rad50wt (-, top). n = 3. e Representative live-cell images of untreated or CPT-treated rad50\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\triangle$$\end{document}? cells expressing Rad50wt/ho/lo-yEVenus with histone Hta2-mCherry as nuclear marker. Arrows indicate nuclear Rad50 foci (Supplementary Fig. 7a). f Anti-Rad50 western blot showing expression levels of Rad50wt/ho/lo-yEVenus in rad50\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\triangle$$\end{document}? cells before (-) and after (+) CPT treatment. a-Tubulin controls equal loading. n = 3. g Quantification of live-cell imaging in e. The percentage of cells with nuclear Rad50-yEVenus foci (arrows in e) is shown for untreated and CPT-treated rad50\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\triangle$$\end{document}? cells expressing Rad50wt/ho/lo-yEVenus. Mean ± SD, n = 3 independent experiments, unpaired two-tailed t-test. Scale bars: 5 µm.
Fig 5: Phosphorylated C-terminus of Sae2 interacts with Rad50. a Full-length phosphorylated recombinant MBP-tagged pSae2 was mock-treated or dephosphorylated with ? phosphatase upon binding to amylose resin and incubated with recombinant MRX complex. The eluates were visualized by silver staining. Prescission protease was added to all samples as a protein stabilizer and to cleave the MBP tag off pSae2. b The FLAG-tagged recombinant Rad50 protein was immobilized on anti-FLAG affinity resin and incubated with phosphorylated C-terminal domain of pSae2 (pSae2 ?N169, residues 170–345), which had been either mock-treated or dephosphorylated with ? phosphatase. The bound proteins were eluted and detected by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. c The phosphorylated recombinant MBP-tagged C-terminal domain of pSae2 (residues 170–345) was bound to amylose resin, eluted, cleaved with prescission protease, and immobilized on NiNTA resin. The bound pSae2 ?N169 was mock-treated or dephosphorylated with ? phosphatase and incubated with recombinant wild-type Rad50 or ATP binding-deficient Rad50 K40A. Proteins were eluted and visualized by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. d Assay as in c. Phosphorylated C-terminal domain of pSae2 was incubated with wild-type Rad50 or Rad50 K81I (representative Rad50S) mutant
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