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Single locus phosphoproteomics reveals phosphorylation of RPA-1 is required for generation of single-strand DNA following a break at a subtelomeric locus

ABSTRACT: Damage to the genetic material of the cell poses a universal threat to all forms of life. Central to the DNA damage response (DDR) is a phosphorylation signalling cascade that leads to the co-ordination of the cellular response to a DNA break. Identifying the proteins that are phosphorylated is crucial to understanding the mechanisms underlying this DDR. We have used SILAC-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following a single double strand break (DSB) at a chromosome internal locus and the subtelomeric bloodstream form expression site in Trypanosoma brucei. We report >6500 phosphorylation sites, including a core set of 211 DSB responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we find that there is a striking distinction between the proteins phosphorylated in response to a chromosome internal DSB and one at the active BES and describe a single phosphorylation event on Replication factor A (RPA) 1 that is required for efficient resection at a bloodstream form expression site

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: MARIETTE MATONDO

LAB HEAD: Lucy Glover

PROVIDER: PXD034455 | Pride | 2024-09-04

REPOSITORIES: pride

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			Action	DRS
	Phospho__STY_Sites.txt	Txt
	Raw1-2INPUT.7z	Other
	Raw1-2Phospho.7z	Other
	Raw3-4INPUT.7z	Other
	Raw3-4Phospho.7z	Other

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DNA Double-Strand Breaks Induce H2Ax Phosphorylation Domains in a Contact-Dependent Manner

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Nuclear poly(A)-binding protein 1 is an ATM target and essential for DNA double-strand break rep

Project description:The DNA damage response (DDR) is an extensive signaling network that is robustly mobilized by DNA double-strand breaks (DSBs). The primary transducer of the DSB response is the protein kinase, ataxia-telangiectasia, mutated (ATM). Here, we establish nuclear poly(A)-binding protein 1 (PABPN1) as a novel target of ATM and a crucial player in the DSB response. PABPN1 usually functions in regulation of RNA processing and stability. We establish that PABPN1 is recruited to the DDR as a critical regulator of DSB repair. A portion of PABPN1 relocalizes to DSB sites and is phosphorylated on Ser95 in an ATM-dependent manner. PABPN1 depletion sensitizes cells to DSBinducing agents and prolongs the DSB-induced G2/M cell-cycle arrest, and DSB repair is hampered by PABPN1 depletion or elimination of its phosphorylation site. PABPN1 is required for optimal DSB repair via both nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR), and specifically is essential for efficient DNAend resection, an initial, key step in HRR. Using mass spectrometry analysis, we capture DNA damage-induced interactions of phospho-PABPN1, including well-established DDR players as well as other RNA metabolizing proteins. Our results uncover a novel ATM-dependent axis in the rapidly growing interface between RNA metabolism and the DDR.

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Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress

Project description:The DNA damage response (DDR) is a complex signaling network that is robustly mobilized by double-strand breaks (DSBs) in the DNA. This network relies on extensive cascades of protein phosphorylation and dephosphorylation, which are initiated by three apical protein kinases that belong to the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR and DNA-PK. Loss of ATM leads to a prototypic genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the DSB-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique selected reaction monitoring (SRM). We uncovered redundant as well as non-redundant roles of the PIKKs following genotoxic stress and obtained evidence of a DNA-PK-dependent attenuation of the ATM response. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics to each of them. Our results shed light on the intricate relationships between the three PIKKs in regulating the DDR.

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2024-02-15 | PXD042607 | Pride

Cohesin profiling at DSBs

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