Rad5 Prevents the Accumulation of ssDNA Gaps at Stressed DNA Replication Forks
Bibliographic record
Abstract
In S. cerevisiae Rad5 is a component of the post‐replication repair pathway (PRR) and possesses both E3‐ubiquitin ligase and DNA helicase activity. We recently found that Rad5 forms sub‐nuclear foci after treatment with hydroxyurea (HU). Previous studies indicate that Rad5‐dependent poly‐ubiquitination of PCNA is required for error‐free bypass of MMS‐ or UV‐induced DNA lesions but there is little insight into the role of Rad5 in response to HU‐induced replication stress, where base lesions are likely absent. Here we define the function of Rad5 during HU‐induced replication stress. Rad5 foci form predominantly during S phase of the cell cycle and are induced by both chemical and genetic replication stress, suggesting that Rad5 foci correspond to Rad5 action at stressed DNA replication forks. Using chromatin immunoprecipitation followed by deep sequencing (ChIP‐seq) we show that Rad5 is recruited and retained at HU‐stressed forks, and that recruitment depends on mono‐ubiquitination of PCNA. To reveal the function of Rad5 at stressed‐forks we monitored cell cycle progression of WT and rad5 null cells recovering from HU‐induced replication stress. In this scenario rad5 null cells delay in G2/M with an activated DNA damage checkpoint, chromatin bridges, and increased RPA foci, indicating the presence of ssDNA. The ssDNA that accumulates in rad5 Δ is not a product of incomplete replication at specific loci, and is not the result of abnormal recombination. In fact, deletion of the anti‐recombinase gene SRS2 rescues the accumulation of chromatin bridges in rad5 Δ, suggesting that activating homologous recombination during S phase can compensate for rad5 deficiency during replication stress. We use a modified version of DNA combing to show the presence of ssDNA gaps directly, and to show that these accumulate in rad5 Δ. Taken together, our results indicate that Rad5 prevents accumulation of ssDNA and allows for non‐recombinogenic recovery from replication stress. Support or Funding Information Funded by the Canadian Cancer Society Research Institute and the Natural Sciences and Engineering Research Council of Canada This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 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 | 0.000 | 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) | 0.002 | 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 source (direct Gemma or distilled Codex), 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".