Abstract A04: Phosphorylation and ubiquitylation on the RPA-ssDNA platform promote homologous recombination
Bibliographic record
Abstract
Abstract Replication stress is an important source of genome instability in cancer cells. Impediments to replication fork progression stimulate the accumulation of RPA-coated single-stranded DNA (RPA-ssDNA). RPA-ssDNA then recruits and activates a large number of genome maintenance factors including the master checkpoint kinase ATR to protect genomic integrity. Amongst the factors recruited onto RPA-ssDNA, we have previously shown that the E3 ubiquitin ligase PRP19, in addition to its central role as an RNA splicing factor, plays an integral part in the elicitation of the DDR during replication stress. Specifically, the ubiquitin ligase activity of PRP19 on the RPA-ssDNA platform is critical for the activation of the ATR checkpoint kinase and promotes replication fork stability during stress. How exactly is PRP19 tethered to RPA-ssDNA in response to damage and whether its ubiquitin ligase activity on this platform is important for specific DNA repair pathways remains unexplored. Here, we show that RPA ubiquitylation is potently triggered by genotoxic agents which target replication forks. The ubiquitylation of RPA and its interaction with PRP19 both correlate with RPA32 phosphorylation. In fact, we show that ubiquitylated RPA is also phosphorylated. Interestingly, a non-phosphorylatable RPA32 mutant interacts poorly with PRP19 and is not efficiently ubiquitylated. Furthermore, we identify a positively charged pocket on the PRP19 WD40 domain which interacts with RPA during replication stress suggesting that PRP19 may recognize phosphorylated RPA through this surface. Finally, we show that the ubiquitin ligase activity of PRP19 and its ability to interact with RPA through its electropositive pocket are both required for optimal homologous recombination. Our work sheds new light on the regulation of RPA-ssDNA ubiquitylation which is critical for genome stability. We show that a splicing factor, PRP19, reallocates its activity in response to damage through the combined activities of PI3K-like kinases on RPA-ssDNA, highlighting the intricate crosstalk between RNA maturation factors and genome maintenance. We propose a model whereby RPA phosphorylation promotes its ubiquitylation by PRP19 and potentially other ubiquitin ligases and favors error-free repair of DNA breaks via homologous recombination. Because of their roles in replication stress tolerance, targeting mediators of RPA ubiquitylation may synergize with chemotherapeutic agents that inhibit DNA replication thereby improving the efficiency of cancer treatments. Citation Format: Jean-Christophe Dubois, Geneviève Clément, Laurent Cappadocia, Luc Gaudreau, Lee Zou, Alexandre Maréchal. Phosphorylation and ubiquitylation on the RPA-ssDNA platform promote homologous recombination [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A04.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".