Dataset Information

Temporal modulation of the NF-kB network in response to different types of DNA damage ETOPOSIDE C4PR_LIV

ABSTRACT: Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to the DNA damage response pathway. Yet, understanding of the co-ordinated signalling mechanisms that result in these different DNA damaging agents inducing specific cellular outcomes through RelA remains unclear. Here, we examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) on the phosphorylation status of RelA and its protein binding partners, using label-free quantitative phosphoproteomics. Although there were relatively few stimulus specific differences overall in the phosphorylated RelA interactome in response to either DNA damaging agent, we observed subtle, but significant changes in the phosphorylation states of the RelA bound proteins as a function of both the type of and duration of the DNA damaging agent used. The DNA double strand break (DSB) inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and (unsurprisingly) DSB repair. Kinase substrate prediction of confident, differentially regulated phosphosites suggests possible roles for CDK1 and MAPK3/ERK1 signaling, in addition to the known roles of ATM/ATR. In contrast, HU-induced replicative stress mediated more temporally dynamic regulation, with phosphoprotein components of the RelA network having known roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif. Our data thus point to differential regulation of key cellular processes and the involvement of unique signalling pathways in modulating DNA damage-specific functions of RelA.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Catarina Franco

LAB HEAD: Claire Eyers

PROVIDER: PXD019587 | Pride | 2021-09-09

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	CF_TiO2_enrich_120MIN_1-01.msf	Msf
	CF_TiO2_enrich_120MIN_1.msf	Msf
	CF_TiO2_enrich_120MIN_1.raw	Raw
	CF_TiO2_enrich_120MIN_2.msf	Msf
	CF_TiO2_enrich_120MIN_2.raw	Raw

Items per page:

1 - 5 of 30

Publications

Temporal modulation of the NF-κB RelA network in response to different types of DNA damage.

Campbell Amy E AE Ferraz Franco Catarina C Su Ling-I LI Corbin Emma K EK Perkins Simon S Kalyuzhnyy Anton A Jones Andrew R AR Brownridge Philip J PJ Perkins Neil D ND Eyers Claire E CE

The Biochemical journal 20210201 3

Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics ...[more]

PMID: 33438746

Similar Datasets

Project description:Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed. Keywords: ChIP-chip â¢ The goal of the experiment Genome-wide localization of Rad52 binding sites in Saccharomyces cerevisiae â¢ Experimental factor Distribution of Rad52 on chromosome III, IV, and V and the right arm of chromosome VI Strain: wild type, rad53 mutant, and rad53 siz2 mutant (W303 background, expressing myc tagged protein from its native promoter) Cell condition 1: G1 arrest with alpha-factor Cell condition 2: HU treatment â¢ Experimental design ChIP analyses: ChIP using anti-c-Myc antibody. ChIP-chip analyses: In all cases, hybridization data of ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F) were used. â¢ Quality control steps taken Confirmation of several loci by quantitative real time PCR. Wild type cells expressing non-tagged Rad52 were used as a negative control of DNA amplification.

Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for DSBs after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 1 h after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, and R 1h, respectively. The strains from which the samples were collected are indicated following the time point, e.g. G1_MEC1 or R 1h_mec1. The experiment with mec1 was done twice (Experiments A and B) and that with MEC1 was done once (Experiment C). In addition, a control experiment of in vitro digestion with BamHI using the G1_mec1 sample (G1_BamHI) was performed.