TRAIP resolves DNA replication-transcription conflicts during the S-phase of unperturbed cells

Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We conclude that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.

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ChIP-seq Data deposition
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Files in database submission
Mouse anti-! Actin loading controls (Santa Cruz sc-47778, C4, HRP-conjugated; 1:5000 in 5% BSA in TBST). Rabbit anti-UBXN7 (Thermo Fisher Scientific 15779771; 1:1000) in 5% Milk in TBST. Rabbit Anti-SPRTN (Novus Biologicals NBP1-84163; 1:1000) in 5% Milk in TBST. The validation of antibodies raised against Xenopus laevis proteins in-house have been described previously in Xenopus egg extract system, and as such are referenced appropriately in the text. Human TRAIP antibodies, were validated in previous manuscripts as cited. all other commercially available antibodies were validated for the assays we used them for and the validation is provided online on the manufactures' websites. For derivatives of the original cells generated during this study the genotype was verified by PCR amplification and sequencing of the relevant loci and the protein levels established by western blotting with the appropriate antibodies.
All cell lines were tested for Mycoplasma using the EZ-PCR Mycoplasma testing kit (Biological Industries). Negative results were confirmed by including a +(ve) control provided by the kit.
No commonly misidentified lines were used in this study.

Xenopus laevis
Provide details on animals observed in or captured in the field; report species, sex and age where possible. Describe how animals were caught and transported and what happened to captive animals after the study (if killed, explain why and describe method; if released, say where and when) OR state that the study did not involve wild animals.
For laboratory work with field-collected samples, describe all relevant parameters such as housing, maintenance, temperature, photoperiod and end-of-experiment protocol OR state that the study did not involve samples collected from the field. Quality of the sequencing was assessed by FASTQC. Peaks were identified according to the parameters above; only peaks identified in both the experimental repeats were further characterized into details.

EaSeq.net
For un-extracted cells, following the required experimental procedures (e.g., proliferation curves) cells were harvested and fixed in 70% ethanol in PBS for 16 hours at -20C. Following fixation, cells were washed twice in washing buffer (5% BSA, 0.1% Tween-20, PBS) and antibody staining carried out if required. Briefly, washed cells were resuspended in 100l primary antibody in washing buffer and incubated at room temperature for 1 hour, rocking to prevent cells from settling. The cells were washed twice in washing buffer and re-suspended in 100l secondary antibody in washing buffer for 1 hour at room temperature in the dark. Stained cells were washed 1X in washing buffer and 2X in D-PBS (-) before being resuspended in either Hoechst Staining Buffer (5g/ml Hoechst 33582, PBS) or Propidium Iodide Staining Buffer (50g/ml Propidium Iodide, 50g/ml RNase A, PBS). For BrdU detection, 10M of BrdU was added to the growth media 1 hour prior to harvesting. Cells were collected and fixed in ethanol as described. Fixed cells were washed once in PBS before being resuspended in 1 ml 2 M HCL supplemented with 0.1 mg/ml Pepsin for 20 minutes. Cells were then washed, and antibody staining carried out as described. To explore the replisome binding pattern on chromatin, cells were extracted using CSK buffer (25 mM HEPES pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM Sucrose, 0.5% Triton X-100, 1X complete protease inhibitors) to remove soluble fractions. The protocol used to extract cells has been described elsewhere (Forment & Jackson, 2015).
All flow cytometry was carried out using a Beckman CytoFlex Instrument.
Software used: Beckman CytExpert V. 2.5. and FLowJo V.10 N/A as we were not isolating specific cells within a population.