Dataset Information

A multi-scale map of protein assemblies in the DNA damage response

ABSTRACT: The DNA damage response (DDR) ensures error-free genome replication and transcription and is disrupted in numerous pathologies including cancer, inflammation and aging1–5. An ongoing challenge is to determine the proteins orchestrating DDR and their organization into protein assemblies and complexes, some of which are constitutive, some which emerge de novo or are remodeled in DNA damaging conditions6. Here we integrate multi-conditional protein interaction networks with diverse multi-omics datasets to create a comprehensive map of DDR protein assemblies at multiple scales of analysis. This DNA damage response assembly map (DDRAM) encompasses 336 proteins, of which 57 (17%) are newly implicated in sensing or repair of DNA damage. We establish a requirement for three such factors in homologous recombination, the lipid chaperone FABP5, the actin-depolymerizing factor GSN, and the galectin LGALS7 as part of the BRCA2/PALB2 DNA repair complex. Another 204 (61%) proteins are known DDR factors assigned more specific functions. We find that single-strand DNA repair (SSR) factors segregate into a collection of 18 nested assemblies, which mirrors the function and dynamic aggregation of these factors at sites of DNA damage. One of these factors, CHTF18, localizes in a PARP-dependent manner with dynamics distinct from those in other SSR assemblies. The map is available at ccmi.org/ddram for interactive visualization, query and download.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Breast Cancer

SUBMITTER: Minkyu Kim

LAB HEAD: Nevan Krogan

PROVIDER: PXD037494 | Pride | 2024-07-03

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	DDRAM_new-baits_AP-MS_MDA231.zip	Other
	MS_new_Baits_noEto_Eto_Sample_Annotations__DDRAM_.xlsx	Xlsx
	checksum.txt	Txt
	qx027073.raw	Raw
	qx027075.raw	Raw

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Publications

A multi-scale map of protein assemblies in the DNA damage response.

Kratz Anton A Kim Minkyu M Kelly Marcus R MR Zheng Fan F Koczor Christopher A CA Li Jianfeng J Ono Keiichiro K Qin Yue Y Churas Christopher C Chen Jing J Pillich Rudolf T RT Park Jisoo J Modak Maya M Collier Rachel R Licon Kate K Pratt Dexter D Sobol Robert W RW Krogan Nevan J NJ Ideker Trey T

Cell systems 20230522 6

The DNA damage response (DDR) ensures error-free DNA replication and transcription and is disrupted in numerous diseases. An ongoing challenge is to determine the proteins orchestrating DDR and their organization into complexes, including constitutive interactions and those responding to genomic insult. Here, we use multi-conditional network analysis to systematically map DDR assemblies at multiple scales. Affinity purifications of 21 DDR proteins, with/without genotoxin exposure, are combined w ...[more]

PMID: 37220749

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Project description:The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1. Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis (Survivin, HIF1α, XIAP), promoting cell cycle arrest (GADD45a, p53, ATRIP, Chk1) and DNA repair (53BP1, BRCA1/2, PARP, Rfc2-5, ATM, MRE-11, others). Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. While some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage. The purpose of this study was to identify mRNAs that are selectively increased in translation by high levels of the initiation factor eIF4G1, which occurs in many advanced human cancers, in response to DNA damage caused by ionizing radiation, Polysome isolation was performed by separation of ribosome-bound mRNAs in sucrose gradients. Beckman Ultra-Clear centrifuge tubes were loaded with 5.5 ml of 50% sucrose in low salt buffer (LSB: 200 mM Tris pH7.4 in DEPC H2O, 100 mM NaCl, 30 mM MgCl2) with 1:1000 RNasin (Fermentas) and 100 μg/mL cycloheximide (CHX) in ethanol and incubated at 4°C horizontally overnight. Media was removed from cell cultures, replaced with media containing 100 μg/mL CHX, cells incubated at 37°C for 10 min to halt protein synthesis, and trypsinized in trypsin/EDTA containing 100 μg/mL CHX. Cells were washed twice in PBS containing CHX, RNasin and Roche complete EDTA-free protease inhibitor tablet, lysed in LSB with CHX and RNasin. Lysates were added to a Dounce homogenizer and incubated on ice for 3 min before addition of Triton detergent buffer (1.2% Triton N-100, 0.2M sucrose in LSB) and homogenization. Samples were transferred to cold sterile Eppendorf centrifuge tubes and centrifuged at 13,000 x RPM for 10 min at 4°C. Post-nuclear supernatants were transferred to centrifuge tubes containing 100 μL Heparin solution (10 mg/mL heparin, 1.5M NaCl in LSB) with RNasin and CHX, applied to a sucrose gradient. Gradients were centrifuged at 36K RPM at 4°C for 2 h, and supernatants recovered with an ISCO-UV fractionator. Samples were recovered into Eppendorf centrifuge tubes containing 40 μL RNase-free 0.5M EDTA and kept on ice. One volume acidic phenol-chloroform was added to each sample, which were then mixed and centrifuged at 10,000 x g for 15 min. The acidic phenol-chloroform extraction was repeated with the aqueous phase, which was then extracted twice via chloroform extraction. RNA was precipitated at -20°C overnight by addition of isopropanol with 0.1 vol NaOAc, pH5.2. The following day, RNA pellets were recovered by centrifugation, washed with 70% ethanol, air-dried and resuspended in sterile H2O. RNA from fractions was then pooled based on their relative rate of translation, with fractions containing 4 or more ribosomes considered heavily translated and used for gene chip analysis. The RNA quality was examined via the Agilent Technologies kit.