scholarly journals The Human Exonuclease-1 Interactome And Phosphorylation Sites

2019 ◽  
Author(s):  
Wassim Eid ◽  
Daniel Hess ◽  
Christiane König ◽  
Christian Gentili ◽  
Stefano Ferrari
Keyword(s):  
Mass Spectrometry ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Rna Processing ◽  
List Type ◽  
Phosphorylation Sites ◽  
Interacting Proteins ◽  
Damage Response ◽  
Causative Agents ◽  
Exonuclease 1

ABSTRACTError-free repair of DNA double-strand break is orchestrated by homologous recombination (HR) pathways and requires the concerted action of several factors. Among these, EXO1 and DNA2/BLM execute extensive resection of DNA ends to produce 3’-overhangs, which are key intermediates for downstream steps of HR. To help shedding light on regulatory aspects of DNA repair pathways in which EXO1 participates, we set out to identify proteins interacting with EXO1. Affinity purification of EXO1 followed by Orbitrap mass spectrometry led to the identification of novel partners that are involved in RNA processing or that are the causative agents of rare X-linked disorders. Depletion of a selected subset of EXO1 interacting proteins led to reduction of the DNA damage response. Among those, we examined the RRP5-homologue and NFκB-interacting protein PDCD11/ALG-4, which has roles in apoptosis and is a putative driver gene in cutaneous T-cell lymphoma. We provide evidence that depletion of PDCD11 decreased the formation of γH2AX foci and the phosphorylation of DNA damage response signaling intermediates in response to camptothecin or bleomycin, resulting in increased cellular resistance to DNA damage. Furthermore, extensive coverage of EXO1 sequence (>85%) by mass spectrometry allowed conducting an in-depth analysis of its phosphorylation sites, with the identification of 26 residues that are differentially modified in untreated conditions or upon induction of DNA damage.As a whole, these results provide the basis for future in-depth studies on novel roles of EXO1 in genome stability and indicate targets for pharmacological inhibition of pathways of cancer development.HIGHLIGHTSProteome-wide analysis of Exonuclease-1 (EXO1) interacting proteins revealed novel partners involved in RNA processing or that are the causative agents of rare X-linked disorders.We provide evidence for a role of PDCD11 in the DNA Damage Response.We conducted a comprehensive identification of EXO1 phosphorylation sites.

scholarly journals The Role of UBR5 Mutations in the Pathogenesis of Mantle CELL Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4124-4124
Author(s):  
Olga Kutovaya ◽  
Stacy Hung ◽  
Hughes Christopher ◽  
Randy D Gascoyne ◽  
Morin Gregg ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Cycle ◽  
Dna Damage ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Interacting Proteins ◽  
Mantle Cell ◽  
Damage Response

Abstract Intro: Mantle cell lymphoma (MCL) accounts for 6% of non-Hodgkin lymphomas and represents a particularly challenging disease with patient outcomes inferior to most other lymphoma subtypes. Using targeted capture sequencing of MCL biopsy samples, we recently reported frequent mutations (18%) in UBR5, a gene encoding an E3 ubiquitin-protein ligase that has not been previously implicated in lymphomagenesis. All mutations were clustered within 100bp in or around exon 58 of UBR5 and truncate the reading frame or change a key lysine residue. These mutations are predicted to result in the loss of the conserved cysteine residue in the HECT-domain, which is responsible for binding the ubiquitin co-factor. The recurrence and clustering of UBR5 mutations suggest their critical pathogenic involvement in a subgroup of MCL that might be therapeutically targetable. The aim of this study is to determine UBR5 mutation-associated proteome changes and altered cell signaling. Methods: As seen in MCL patients, mutations in exon 58 of UBR5 were introduced to three MCL cell lines (Granta-519, Jeko-1, and Mino) using the CRISPR-Cas9 genome engineering tool. First, mass spectrometry-based immunoprecipitation proteomics (IP-MS) was employed to identify differences in UBR5 interacting partners between UBR5 mutant and wildtype (WT) cells. Candidate UBR5 interacting proteins were validated by flow cytometry, western blotting, co-immunoprecipitation, and immunofluorescence. Next, global proteomes of UBR5 mutants and WT were analyzed by Tandem Mass Tag (TMT)-based mass spectrometry quantification to identify proteins with differential expression due to the UBR5 mutations. Results: The IP-MS analysis of WT vs UBR5 mutants revealed histone and cell cycle control proteins as candidate differential UBR5 interacting proteins (p<0.05). Particularly, histones H1, H4, and H2AFX, as well as the cell cycle genes CDC5L, BUB3, MAP4, RAD50 and CDK11B were identified as candidate UBR5 interacting partners. The global proteome analysis identified a set of differentially expressed genes (mutant vs wt; p<0.05) that are common among the MCL cell lines with the same direction of change. Gene ontology analysis of this set revealed DNA damage response, chromosome organization, and cell cycle response pathways as the predominant pathways affected. Moreover, our preliminary functional studies indicate constitutive phosphorylation of H2AFX in UBR5 mutants vs WT in line with the role of UBR5 in DNA damage response. Conclusions: Our results are consistent with UBR5 functioning as a key regulator of cell signalling and strongly suggest UBR5 as a novel regulator of histone modifications and DNA damage response. These findings provide an experimentally valid platform for further functional investigation and testing of target therapies for MCL harbouring UBR5 mutations. Disclosures No relevant conflicts of interest to declare.

scholarly journals The RNA Processing Factor Y14 Participates in DNA Damage Response and Repair

iScience ◽  
2019 ◽  
Vol 13 ◽  
pp. 402-415 ◽  
Author(s):  
Tzu-Wei Chuang ◽  
Chia-Chen Lu ◽  
Chun-Hao Su ◽  
Pei-Yu Wu ◽  
Sarasvathi Easwvaran ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Rna Processing ◽  
Processing Factor ◽  
Damage Response

scholarly journals PARprolink: a photoaffinity probe for identifying poly(ADP-ribose)-binding proteins

2020 ◽  
Author(s):  
Morgan Dasovich ◽  
Morgan Q. Beckett ◽  
Scott Bailey ◽  
Shao-En Ong ◽  
Marc M. Greenberg ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Rna Processing ◽  
Binding Proteins ◽  
Post Translational Modification ◽  
Mobility Shift ◽  
Binding Partners ◽  
Damage Response ◽  
Prostate Cancers ◽  
Electrophoretic Mobility Shift Assays

ABSTRACTPost-translational modification of proteins with poly(ADP-ribose) (PAR) is an important component of the DNA damage response. Four PAR synthesis inhibitors have recently been approved for the treatment of breast, ovarian, and prostate cancers. Despite its clinical significance, a molecular understanding of PAR function, including its binding partners, remains incomplete. In this work, we synthesize a PAR photoaffinity probe that captures and isolates endogenous PAR binders. Our method identified dozens of known PAR-binding proteins and hundreds of novel binders involved in DNA repair, RNA processing, and metabolism. PAR binding by eight candidates was confirmed using pull-down and/or electrophoretic mobility shift assays. Using PAR probes of defined lengths, we detected proteins that preferentially bind to 40-mer over 8-mer PAR, indicating that polymer length may regulate the outcome and timing of PAR signaling pathways. This investigation produces the first census of PAR-binding proteins, provides a proteome-wide view of length-selective PAR binding, and associates PAR binding with RNA metabolism and the formation of biomolecular condensates.

scholarly journals The RNA processing factors THRAP3 and BCLAF1 promote the DNA damage response through selective mRNA splicing and nuclear export

2017 ◽  
Vol 45 (22) ◽  
pp. 12816-12833 ◽  
Author(s):  
Jekaterina Vohhodina ◽  
Eliana M. Barros ◽  
Abigail L. Savage ◽  
Fabio G. Liberante ◽  
Lorenzo Manti ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Rna Processing ◽  
Nuclear Export ◽  
Mrna Splicing ◽  
Damage Response ◽  
Processing Factors

scholarly journals ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function

Blood ◽  
2009 ◽  
Vol 113 (10) ◽  
pp. 2181-2190 ◽  
Author(s):  
Natalie B. Collins ◽  
James B. Wilson ◽  
Thomas Bush ◽  
Andrei Thomashevski ◽  
Kate J. Roberts ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Posttranslational Modifications ◽  
S Phase ◽  
Damage Response ◽  
Pathway Function

Abstract Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage–specific event that is downstream of ATR and is functionally important in the FA pathway.

scholarly journals Proteomic Investigations Reveal a Role for RNA Processing Factor THRAP3 in the DNA Damage Response

2012 ◽  
Vol 46 (2) ◽  
pp. 212-225 ◽  
Author(s):  
Petra Beli ◽  
Natalia Lukashchuk ◽  
Sebastian A. Wagner ◽  
Brian T. Weinert ◽  
Jesper V. Olsen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Rna Processing ◽  
Processing Factor ◽  
Damage Response

scholarly journals The DNA damage response activates HPV16 late gene expression at the level of RNA processing

2018 ◽  
Vol 46 (10) ◽  
pp. 5029-5049 ◽  
Author(s):  
Kersti Nilsson ◽  
Chengjun Wu ◽  
Naoko Kajitani ◽  
Haoran Yu ◽  
Efthymios Tsimtsirakis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Rna Processing ◽  
Late Gene ◽  
Late Gene Expression ◽  
Damage Response
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