Phosphoproteomics sample preparation impacts biological interpretation of phosphorylation signaling outcomes

The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods-strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled by both methods achieved equivalent coverage (around 20,000 phosphosites), whereas a combined dataset significantly increased the depth (>30,000 phosphosites). While both methods reproducibly quantified a subset of shared IR-responsive phosphosites that represent DNA damage and cell-cycle-related signaling events, most IR-responsive phosphoproteins (>82%) and phosphosites (>96%) were method-specific. Both methods uncovered unique insights into phospho-signaling mediated by single (SCXPhos) versus double/multi-site (HighPhos) phosphorylation events; particularly, each method identified a distinct set of previously unreported IR-responsive kinome/phosphatome (95% disparate) directly impacting the uncovered biology.Genome Instability and Cance

Transcription-coupled nucleotide excision repair is coordinated by ubiquitin and SUMO in response to ultraviolet irradiation

Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitin-like modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation

TRiC controls transcription resumption after UV damage by regulating Cockayne syndrome protein A

Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA-DDB1-CUL4A-RBX1 cullin-RING ubiquitin ligase complex (CRLCSA). Despite its vital role in TC-NER, little is known about the regulation of the CRLCSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin. TRiC's binding to CSA ensures its stability and DDB1-dependent assembly into the CRLCSA complex. Consequently, loss of TRiC leads to mislocalization and depletion of CSA, as well as impaired transcription recovery following UV damage, suggesting defects in TC-NER. Furthermore, Cockayne syndrome (CS)-causing mutations in CSA lead to increased TRiC binding and a failure to compose the CRLCSA complex. Thus, we uncover CSA as a TRiC substrate and reveal that TRiC regulates CSA-dependent TC-NER and the development of CS

Solar UV damage to cellular DNA: from mechanisms to biological effects

Genome Instability and Cance

Isolation and characterization of a mammalian nuclear DNA-protein complex

Contains fulltext : mmubn000001_025237950.pdf (publisher's version ) (Open Access)Promotor : C. Kuyper120 p

Nucleotide excision repair: damage recognition, complex assembly and its regulation

Toxicogenetic

Transcription factor IIS impacts UV-inhibited transcription

Inhibition of transcription elongation can cause severe developmental and neurological abnormalities notably manifested by the rare recessive progeroid disorder Cockayne syndrome (CS). DNA alterations can cause permanent blocks to an elongating RNA polymerase II (RNAPII) leading to transcriptional arrest. Abrogation of transcription arrest requires removal of transcription blocking lesions through transcription-coupled nucleotide excision repair (TC-NER) a process defective in CS. Transcription elongation factor IIS (TFIIS) has been found to localize with the TC-NER complex after cellular exposure to UV-C light and in vitro addition of TFIIS to a damage arrested RNAPII causes transcript shortening. Hence default TFIIS activity might mimic or contribute to the severe phenotype of Cockayne syndrome. Here we show that down regulation of TFIIS by siRNA treatment of human cells lead to impaired RNA synthesis recovery and elevated levels of hyper-phosphorylated RNAPII after UV-irradiation. TFIIS knock down does not affect TC-NER, the reappearance of hypo-phosphorylated RNAPII post-UV-irradiation, UV sensitivity or the p53 damage response. These findings reveal a role for TFIIS in transcription recovery and re-establishment of the balance between hypo- and hyper-phosphorylated RNAPII after DNA damage repair. (C) 2010 Elsevier B.V. All rights reserved.DNA repair mechanism