Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane
Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane.
Sample Processing Protocol
Myc-tagged Rif1NTD was reduced with TCEP, to reduce disulfide bridges formed by cysteine residues, leaving S-acylation intact. Final concentrations of 50 mM NEM and 0.5% Triton X-100 were added to the cleared lysates, and blocking of reactive cysteines was performed for 2 h at 4 °C on a rotating wheel. Chloroform-methanol precipitates were solubilized in resuspension buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 2% SDS, 8 M urea). Removal of S-acyl groups from cysteines was achieved by incubating the samples with beads buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.1% Triton X-100) containing 10 mM 1,4-Dithiothreitol for 1 h at room temperature. Proteins were precipitated, resuspended, and beads buffer containing 20 mM 2-chloroacetamide was added, allowing CAM-labeling of freed-up cysteines. Myc-tagged Rif1NTD immunoprecipitation was performed overnight at 4 °C by addition of pre-washed anti-Myc-coupled magnetic agarose beads (Chromotek, ytma-20). After extensive washing with high (50 mM Tris-HCl pH 7.5, 500 mM NaCl) and low salt (50 mM Tris-HCl pH 7.5, 150 mM NaCl) buffers, samples were subjected to tryptic digestion on beads and analyzed by mass-spectrometry. Mass spectrometric (PRM) analysis of synthetic peptides Peptides were loaded in 0.1% formic acid, 10% acetonitrile in water onto a 50 um x 15 cm ES801 column (C18, 2 um, 100 Å) and a linear gradient of 2-6% buffer B in buffer A in 2 min, followed by an linear increase from 6 to 30% in 30 min, 30-50% in 10 min, 50-80% in 1 min, and finally the column was washed for 13 min at 80% buffer B at a flow rate of 150 nl/min (buffer A: 0.1% formic acid, 10% acetonitrile in water; buffer B: 0.1% formic acid in acetonitrile). One MS spectrum at 120’000 resolution was acquired from 400-1200 Da, followed by 9 PRM spectra. m/z z t start (min) t stop (min) Name Maximum Injection Time (ms) 725.4 2 18 26 LPLNSYDSANLDK 20 789.9 2 24 32 NDSSLVNFNIQISK 20 809.4 2 27 33 DQTHLESFSSLILK 20 889.9 2 32 39 IENGDDDYILELLEK 20 979.5 2 35 45 TSLPGNPELFSGLLPFLR 20 1041.5 2 20 34 IYQC(CAM)IMLSPVC(CAM)ETIPEK 500 1075.1 2 28 38 IYQCIMLSPVCETIPEK (1Nem/1Cam) 500 1109.1 2 30 40 IYQC(Nem)IMLSPVC(Nem)ETIPEK 200 An isolation window of 1.6 Da, a resolution of 120’000, and an automatic gain control value of 5e4 was used. Fragmentation was performed with a higher energy collision dissociation (HCD) collision energy of 30 eV, and MS/MS scans were acquired with a scan range of 100 to 2000 Da with a resolution of 120’000. Peptides were separated at a flow rate of 300 nl/min with a linear gradient of 10-60% buffer B in buffer A in 10 min, followed by a linear increase from 60 to 90% over 1 min, and the column was finally washed for 5 min at 90% buffer B (buffer A: 0.1% formic acid, 10% acetonitrile in water; buffer B: 0.1% formic acid in acetonitrile). The column was mounted on an Easy ion source connected to an Orbitrap Fusion mass spectrometer (Thermo Scientific).
Data Processing Protocol
PRM data analysis and MASCOT searches PRM data were processed using Skyline 4.1. To compare the four almost identical peptides IYQCIMLSPVCETIPEK with the Cysteins either as NEM or CAM, the same 5 transitions (y3, y9, y10, y11 and y12) were integrated and summed. Mascot v. 2.5 (Matrix Science Ltd.) was used in the Decoy mode to search the Swissprot yeast version 2017_04 including common contaminants. The enzyme specificity was set to trypsin, allowing for up to one incomplete cleavage site. Modification of cysteines with carbamidomethyl (CAM; +57.0245 Da), N-ethylmaleimide (NEM; +125.0477Da), oxidation of methionine (+15.9949 Da), and acetylation of the protein N-terminus (+42.0106 Da) were set as variable modifications. Parent ion mass tolerance was set to 5 ppm and fragment ion mass tolerance to 0.01 Da.
Daniel Hess, Friedrich Miescher Institute for Biomedical Research
Ulrich Rass, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9RQ, United Kingdom ( lab head )